title: Spatially defined InsP3-mediated signaling in embryonic stem cell-derived cardiomyocytes.
abstract: The functional role of inositol 1,4,5-trisphosphate (InsP3) signaling in cardiomyocytes is not entirely understood but it was linked to an increased propensity for triggered activity. The aim of this study was to determine how InsP3 receptors can translate Ca(2+) release into a depolarization of the plasma membrane and consequently arrhythmic activity. We used embryonic stem cell-derived cardiomyocytes (ESdCs) as a model system since their spontaneous electrical activity depends on InsP3-mediated Ca(2+) release. [InsP3]i was monitored with the FRET-based InsP3-biosensor FIRE-1 (Fluorescent InsP3 Responsive Element) and heterogeneity in sub-cellular [InsP3]i was achieved by targeted expression of FIRE-1 in the nucleus (FIRE-1nuc) or expression of InsP3 5-phosphatase (m43) localized to the plasma membrane. Spontaneous activity of ESdCs was monitored simultaneously as cytosolic Ca(2+) transients (Fluo-4/AM) and action potentials (current clamp). During diastole, the diastolic depolarization was paralleled by an increase of [Ca(2+)]i and spontaneous activity was modulated by [InsP3]i. A 3.7% and 1.7% increase of FIRE-1 FRET ratio and 3.0 and 1.5 fold increase in beating frequency was recorded upon stimulation with endothelin-1 (ET-1, 100 nmol/L) or phenylephrine (PE, 10 µmol/L), respectively. Buffering of InsP3 by FIRE-1nuc had no effect on the basal frequency while attenuation of InsP3 signaling throughout the cell (FIRE-1), or at the plasma membrane (m43) resulted in a 53.7% and 54.0% decrease in beating frequency. In m43 expressing cells the response to ET-1 was completely suppressed. Ca(2+) released from InsP3Rs is more effective than Ca(2+) released from RyRs to enhance INCX. The results support the hypothesis that in ESdCs InsP3Rs form a functional signaling domain with NCX that translates Ca(2+) release efficiently into a depolarization of the membrane potential.
authors.affiliation: Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, United States of America.
authors.forename: Nidhi
authors.initials: N
authors.lastname: Kapoor
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois, United States of America.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Maxwell
authors.affiliation: Department of Cell and Molecular Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinois, United States of America.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Mignery
authors.affiliation: Center for Cardiovascular Research, Dept. of Medicine, Section of Cardiology, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: David
authors.initials: D
authors.lastname: Will
authors.affiliation: Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois, United States of America.
authors.forename: Lothar A
authors.initials: LA
authors.lastname: Blatter
authors.affiliation: Center for Cardiovascular Research, Dept. of Medicine, Section of Cardiology, University of Illinois at Chicago, Chicago, Illinois, United States of America.
authors.forename: Kathrin
authors.initials: K
authors.lastname: Banach
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004815:Epidermal Growth Factor; D015870:Gene Expression; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C464944:Emr4 protein, mouse; D053496:Inositol 1,4,5-Trisphosphate Receptors; D004815:Epidermal Growth Factor; D017853:Phosphotransferases (Alcohol Group Acceptor); C051038:Inositol 1,4,5-trisphosphate 3-kinase; D002118:Calcium