Sarah K. England - US grants

This is a starter grant for BIO Postdoctoral Fellowship DBI 94-06860.

DESCRIPTION (provided by applicant): The transition from uterine quiescence to contraction is vital to the health of a newborn and mother, but timing of this event often fails to occur properly; in the U.S., 12% of babies are born preterm, and 20% are delivered following artificial induction of labor. Thus, understanding the regulation of myometrial smooth muscle cell (MSMC) electrical activity and its effect on contraction is essential for both comprehending normal labor and treating dysfunctional labor. Maintenance of uterine quiescence requires an intricate balance between excitatory depolarizing stimuli that promote contractions and inhibitory repolarizing currents that suppress uterine contraction. One predominant channel in MSMCs, the large conductance Ca2+-activated K+ channel (KCa1.1) contributes to quiescence by eliciting a potent repolarizing current in response to excitatory signals, thereby dampening MSMC contraction. In spite of strong evidence supporting the notion that the KCa1.1 channel modulates uterine excitability, the basic mechanisms involved in its physiological regulation during pregnancy remain largely uncharacterized. The long-term goal of my research is to identify the ionic mechanisms that regulate the transition from quiescence to contraction during pregnancy. The objective of this proposal is to define the mechanisms by which the KCa1.1 channel is modulated during pregnancy to control myometrial excitability. Our central hypothesis is that this channel is dynamically modulated by both intrinsic properties and by its association with modulatory proteins. In support of this idea, our preliminary studies in human MSMCs indicate that KCa1.1 is regulated by alternative translation initiation, resulting in KCa1.1 isoforms that vary in their extracellular N- termini. These N-terminal variants differ in their regulation by accessory 1-subunits. We also have generated proteomics data demonstrating that other novel modulators, including the recently described family of - subunits and the protease inhibitor 2macroglobulin (A2M), selectively associate with myometrial KCa1.1 and potentially modify channel activity. The goals of this project are to: 1) define the spatial and temporal interactions between novel proteins that interact with KCa1.1 in non-laboring and laboring human myometrium, 2) identify intrinsic properties of KCa1.1 that alter its association with interacting partners; and 3) determine the mechanism of functional regulation of KCa1.1 in both myometrial cell lines and non-laboring and laboring human myometrium. The research proposed here will establish the molecular pathways that regulate KCa1.1 activity, providing a biological basis for therapies designed to modulate uterine excitability.

I am currently a tenure-track Assistant Professor in the Department of Physiology and Biophysics at the University of Iowa. Presently, I am at a stage of my career development where it is essential to enhance and foster my research program with the long-term goal of obtaining a tenured position at the University of Iowa. My research interest has been in the role of potassium channels in regulating vascular smooth muscle and cardiac myocyte membrane excitability. Recently, I have ventured into the reproductive physiology research area to investigate the role of the large-conductance calcium-activated potassium channel (BKCa) in modulating uterine smooth muscle excitability and contractility during gestation. Potassium channels, due to their ability to potently buffer cell excitation, have been suggested as one potential class of targets for tocolytic therapy. The objective of this proposal is to determine whether modulation of BKCa channel splice variant expression or subunit association correlates to a functional difference in uterine excitability during gestation. The specific aims of this proposal are to: 1) compare transcript and protein expression patterns of BKCa channel isoforms in mouse uterine smooth muscle during gestation, 2) elucidate BKCa channel beta subunit transcript and protein expression during gestation and detemine whether its assembly with the alpha subunit is modulated during pregnancy, 3) determine the contribution of BKCa channel splice variants to the regulation of uterine smooth muscle contraction during gestation, and 4) characterize the expression of the splice variants of the BKCa channel alpha subunit following stimulation with estrogen and progesterone. The University of Iowa provides an excellent environment to complete studies of this nature. The Department of Physiology and Biophysics and University of Iowa College of Medicine has well-established interactive investigators in the fields of ion channel regulation, smooth muscle function, and reproductive endocrinology. With successful attainment of this award, I would be able to devote greater than 80 percent to my research program and familiarizing myself with the literature, research, and investigators in the reproductive sciences field through reading and attending scientific meetings in this area. An Independent Scientist Award will have a major impact in promoting my career development at the University of Iowa.

uterus; potassium channel; birth; molecular biology; muscle contraction; protein structure function; cellular polarity; premature labor; myometrium; gene expression; muscle cells; clinical research;

DESCRIPTION (provided by applicant): In the last decade the University of Iowa has graduated only 38 underrepresented minority M.S. and Ph.D.s, even though the percentages of minorities graduating with bioscience B.S. degrees are similar to non-minorities. We hypothesize that students are not appropriately prepared and/or informed for entrance into competitive postgraduate degree bioscience programs. To address this, the overall goal of the University of Iowa Initiative for Minority Student Development, the Iowa Biosciences Advantage (IBA) program, is to effectively recruit and retain underrepresented minorities aiming to matriculate into biomedical research training programs leading to Ph.D.s, Ph.D./M.D., D.D.S., Ph.D., or M.P.H./Ph.D.s. The breadth and strength of research programs within the biomedical research community of the University of Iowa provides an enriched setting to expose students early in their undergraduate careers to the myriad of opportunities available in the participating colleges. Our specific aims are: 1) to improve diversity of gifted URM students interested in bioscience graduate careers, over and above state means. We will also recruit from within the University of Iowa, focusing on URM candidates already in research laboratories, 2) assist students in making successful high school-first year transitions by providing entering URM students with diverse program initiatives designed to develop students' knowledge, skills, and attitudes, as well as their personal identity, 3) to expose URM biosciences majors to mentored research to promote interest in bioscience Ph.D. programs. We will foster mentoring relationships between IBA students and successful biomedical research faculty, and 4) to provide professional and leadership opportunities to enhance progression into graduate and leadership positions. To achieve these objectives requires the concerted efforts of IBA staff, the Admissions Office, undergraduate faculty, academic advisors, student support services, and support of the participating colleges and departments.

[unreadable] DESCRIPTION (provided by applicant): The Iowa Biosciences Advantage (IBA) is the University of Iowa's Initiative for Maximizing Student Diversity (IMSD) program. The IBA's goal is to identify academically talented undergraduate underrepresented minority students with aspirations for a research career and provide them with first-rate training that will facilitate their entry into doctoral programs in the biomedical, behavioral, and biophysical sciences. Since the last renewal, the IBA program has implemented successful initiatives to facilitate students' transition in college. IBA students conduct year-round mentored research in productive laboratories and gain extensive professional development to aid their competitiveness and progression into graduate school. Since 2003, the percentage of IBA graduates matriculating into doctorate programs has increased from 0% to 11%. Still, the proportion of students who are Ph.D. bound remains low. The main difficulty lies in the identification of high school and undergraduate students with aspirations for careers in research versus the health professions. [unreadable] [unreadable] In the proposed project period we will focus on improving the selection of candidates for bioscience research careers while enhancing the aspects of our program proven successful by comprehensive evaluation. In specific aim 1, IBA will work with current University of Iowa programs to outreach to high schools, community colleges, and current undergraduates. The IBA will execute a Candidate/Scholar two-tier program. New students will be Candidates and gain research experience via laboratory rotations. Scholar status will be awarded competitively to those students in good research and academic standing who intend to pursue doctorate programs upon graduation. With this new system, we will identify students who are research oriented. Specific aim 2 will build on IBA's current successful four-year comprehensive student development curriculum by adding new topics to facilitate matriculation at the University of Iowa and admission to doctoral programs nationally. Specific aim 3 will foster students' career development related specifically to graduate education, research careers, and academia by providing mentored research, partnering with T32 training programs, offering specialized courses to develop students' understanding of the life of a biomedical researcher, offering individualized career planning, and providing opportunities for Scholars to present their research. The last aim will be to facilitate effective mentoring relationships between students and faculty by developing and providing guidelines for IMSD mentors to utilize in the progression of IBA scholars toward the Ph.D. Utilizing faculty and staff expertise in outreach, selection, first-year college experience, student development, career development, graduate education, research funding and responsible conduct of research, mentoring, and evaluation, the IBA will benchmark success with at least 60% of IBA graduates going directly into doctoral programs. [unreadable] [unreadable] The University of Iowa Initiative for Maximizing Student Diversity (IMSD) is an undergraduate program that contributes to the national effort of increasing the numbers of underrepresented minority faculty, investigators and students engaged in biomedical and behavioral research. The program introduces participants to research as early as their first year of college and matches them with faculty research mentors to conduct research throughout their undergraduate tenure. This program provides ongoing and intense academic and research support in an effort to increase the likelihood and rate of matriculation among underrepresented minorities to doctoral research programs. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Despite the fact that oxytocin is used safely to induce or augment labor in the majority of births in the United States, roughly half of all paid obstetric malpractice cases involve claims of its misuse, and the Institute for Safe Medication Practices lists oxytocin as a High-Alert medication. Clinical guidelines for safe use of low versus high doses of oxytocin have recently been published, but the authors recognize the paucity of evidence to support firm recommendations and acknowledge that individual patients may require higher doses than their proposed guidelines. Development of the most effective methods of therapy will require detailed characterization of the basic mechanisms underlying oxytocin's mode of action. Although effects of oxytocin on uterine contractile strength are well documented, multiple studies have now indicated its ability to also regulate the frequency of contraction via an increase in the generation of uterine myometrial smooth muscle cell (MSMC) action potentials; however, the mechanism by which this occurs remains unclear. Intriguingly, oxytocin can depolarize vagal neurons by generating an inward Na+ current that is Na+-dependent and insensitive to the voltage-gated Na+ channel blocker tetrodotoxin. Recently, a sodium leak channel (NALCN; Na+ leak channel, non-selective) with similar properties was identified in MSMCs. Our preliminary studies demonstrate that human MSMCs express NALCN and produce a NALCN-like current, and that inhibition of this channel alters the frequency of human uterine contractions in an ex vivo model. We have also discovered that oxytocin increases NALCN-like current in myometrial cells derived from pregnant women, but not in myometrial cells derived from non-pregnant women. Lastly, oxytocin receptor variants that have attenuated ligand binding have been identified and may affect the response of the uterus to oxytocin either directly or indirectly via NALCN. The objective of this proposal is to advance knowledge of the underlying mechanism of oxytocin action in pregnant women. Both oxytocin and its receptor are important to the process of labor, yet why oxytocin elicits an unpredictable response in women who experience labor arrest is unknown. Recently, identified variants in the oxytocin receptor that have weaker oxytocin binding have been identified, but whether this translates into a clinical presentation of labor protraction or arrest is unknown. Our central hypothesis is that oxytocin binding to the oxytocin receptor regulates the NALCN channel, which underlies the background leak current that sets the frequency of spontaneous rhythmic contractions of the uterus. We speculate that women who require higher doses of oxytocin harbor sequence variants in the oxytocin receptor and will present with altered frequency in their contraction patterns.

? DESCRIPTION (provided by applicant): Proper timing of delivery is important to the immediate and life-long health of both the newborn and the mother, but this event is often mistimed; in the U.S., approximately 12% of babies are born prematurely and up to 10% of pregnancies are described as post-term. For most of pregnancy, the uterus is maintained in a quiescent, non-contractile state in which the myometrial smooth muscle cells (MSMCs) are hyperpolarized, non-excitable, and quiescent. At term, the MSMCs become depolarized, excitable, and contractile. Currently, our limited understanding of how this transition is controlld hampers our ability to treat dysfunctional labor. Numerous ion channels are expressed in the MSMCs and contribute to regulation of uterine excitability. In particular, K+ channels play an important role in maintaining quiescence by controlling MSMC membrane potential by hyperpolarizing the membrane. Another key regulator in control of MSMC excitability is the hormone oxytocin, which binds to the oxytocin receptor (OTR), a G?q-coupled G-protein coupled receptor (G?qCR). As a result, Protein Kinase C (PKC) is activated and Ca2+ is released from intracellular stores, causing activation of actomyosin contraction. Additionally, it has been proposed that oxytocin triggers Ca2+ influx through voltage-dependent calcium channels by depolarizing the MSMC plasma membrane. However, the molecular mechanism responsible for this depolarization has not been established. Here, we propose to test the central hypothesis that the sodium-activated K+ channel SLO2.1 plays a key role in controlling the resting membrane potential of MSMCs and that its activity is down-regulated at term by either oxytocin-mediated inhibition or decreased expression, resulting in membrane depolarization. Several lines of evidence support this hypothesis. First, our preliminary data indicate that SLO2.1 is expressed in human MSMCs. Second, we report that SLO2.1 activity is modulated by oxytocin in both heterologous systems and MSMCs. Finally, SLO2.1 is known to be regulated by G?qCRs. The goals of this projects are the 1) define the temporal and spatial distribution of SLO2.1 channels in MSMCs, 2) investigate modulation of SLO2.1 channels by oxytocin; and 3) assess the contribution of SLO2.1 channels to regulation of uterine contractility. The research proposed here will establish the molecular pathways that regulate SLO2.1 activity, providing a biological basis for therapies designed to modulate uterine excitability.

PROJECT SUMMARY The annual Society for Reproductive Investigation (SRI) meeting brings together both clinical and basic scientists from around the world to discuss research related to women's health and reproductive science. The highly successful annual SRI meeting has had an average attendance of 1164 investigators over the last four years and has brought together established senior and junior investigators to report and discuss their findings in an atmosphere conducive to frank yet amicable exchange. This application seeks funding to cover travel costs to allow 10 trainees and new investigators to attend SRI annual meetings. This proposal also seeks funding to support the travel costs of a senior US-based investigator who will present an invited Distinguished Presidential lecture at the meeting. Meetings are scheduled in March of each year in San Diego, CA (2018), Paris, France (2019), Vancouver, Canada (2020), and Boston, MA (2021). The four-day meeting includes two or three Distinguished Presidential Lectures, oral and poster presentations, 12 mini-symposia, a new investigator plenary, career development and diversity forums, and networking events. The 65th Annual SRI Scientific Meeting, entitled ?Power of Collaboration?, will be held in San Diego, CA, March 7-10. The SRI will continue its efforts to ensure adequate representation of underrepresented minorities (17% in 2017) and women (55% in 2017) as attendees, speakers, and session chairs at each annual meeting.

PROJECT SUMMARY Oxytocin is administered to approximately one-half of the four million women who give birth in the United States each year. A significant challenge for providers is that the oxytocin dose required to induce or augment labor varies by up to 20-fold, and they have no way to predict how, or even whether, a woman will respond to a given dose. This lack of predictability raises important safety concerns and underlies oxytocin's association with adverse maternal events and neonatal outcomes. Thus, it is essential to develop a method to predict oxytocin responsiveness and thereby personalize the dosing regimens. This proposal takes the first step in addressing this need by testing the central hypothesis that the oxytocin responsiveness of uterine (myometrial) smooth muscle cells (MSMCs) can be predicted by oxytocin receptor (OXTR) gene variants. Such variants are common; the Exome Aggregation Consortium identified 132 missense single nucleotide variants (mSNVs) in OXTR, of which ~50% are predicted by mutation analysis software to be deleterious to OXTR function. Our hypothesis is supported by two studies identifying rare mSNVs and common noncoding single nucleotide polymorphisms (SNPs) in OXTR that are associated with oxytocin dose requirement. Additionally, several OXTR coding and noncoding variants have been implicated in adverse reproductive outcomes including preterm birth and long labor duration. Although these studies provide evidence that OXTR variants associate with clinically important phenotypes, the underlying mechanisms are unknown. This lack of knowledge hampers our ability to translate OXTR genetics to personalized labor management approaches. To fill this gap, we propose to determine the effects of mSNVs and common SNPs on OXTR expression and function in MSMCs by pursuing the following Specific Aims: 1) Determinw the mechanisms by which OXTR mSNVs affect oxytocin signaling, 2) Determine the effect of OXTR noncoding SNPs on OXTR mRNA and protein expression in MSMCs, and 3) Developing and test a computational model to predict the effect of OXTR variants on oxytocin signaling efficacy. The work proposed here will be directed under a multi-PI plan bringing together Dr. Sarah England, who has expertise in reproduction and myometrial smooth muscle, and Dr. Princess Imoukhuede, who uses quantitative and computational approaches to define the cellular and molecular underpinnings of disease and has specific expertise in quantitative analysis of receptors. Successful completion of these aims will provide important information regarding the influence of OXTR variants on responsiveness to oxytocin.

PROJECT SUMMARY In the US, nearly half of all pregnant women are treated with a synthetic version of the neuropeptide oxytocin to induce or augment labor or prevent postpartum hemorrhage. However, women require a wide range of oxytocin doses to elicit an appropriate degree of uterine contractility. Furthermore, the oxytocin receptor (OXTR) can cease to respond to oxytocin (become desensitized) after long-term oxytocin administration, often leading to the need for cesarean delivery. Failure of OXTR activation can lead to substantial maternal morbidity and, in extreme cases, mortality due to postpartum hemorrhage. Our long-term goal is to develop strategies to improve OXTR responsiveness and thereby improve safety for pregnant women. In our parent grant, we hypothesized that OXTR genetic variants are contributing to the observed differences in individual response to oxytocin and propose to quantitate these effects in order to build a computational model able to predict response. In this supplement, we propose to develop a new approach for regulation of OXTR activity by identifying and characterizing OXTR positive allosteric modulators (PAMs). OXTR PAMs would be ideal therapeutics because they do not activate their target receptor in the absence of an agonist. Instead, they enhance endogenous ligand activity by binding to the receptor outside of the native ligand binding site. Thus, OXTR PAMs would predominantly enhance OXTR activation in the uterus, where oxytocin concentration is highest, and follow the same temporal OXTR activation pattern brought about by pulsatile oxytocin release during labor. We will pursue two Specific Aims: 1) Identify and validate positive allosteric modulators of OXTR, and 2) Determine how the top hit compounds enhance OXTR signaling. In order to identify the PAMs, we will perform a large-scale screen of diverse small-molecule compounds. In characterizing PAM effects on OXTR signaling, we will also obtain quantitative data to enhance our computational approaches. Together, data from the funded R01 and this supplement will lead to individualized oxytocin administration and an alternative pharmacologic approach to activate OXTR to induce or augment labor.