title: 9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes.
abstract: It is well established that intracellular calcium ([Ca2+]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca2+ clock" controls the chronotropic state of the heart. Recent findings describe a calcium-activated nonselective cation channel (NSCCa) in various cardiac preparations sharing hallmark characteristics of the transient receptor potential melastatin 4 (TRPM4). TRPM4 is functionally expressed throughout the heart and has been implicated as a NSCCa that mediates membrane depolarization. However, the functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. Here, we show by Fura2 Ca-imaging that pharmacological inhibition of TRPM4 in HL-1 mouse cardiac myocytes by 9-phenanthrol (10 μM) and flufenamic acid (10 and 100 μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL-1 cells in Ca(2+)-free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 μM). These pharmacologic agents also depolarize HL-1 cell mitochondrial membrane potential. Furthermore, by on-cell voltage clamp we show that 9-phenanthrol reversibly inhibits membrane current; by fluorescence immunohistochemistry we demonstrate that HL-1 cells display punctate surface labeling with TRPM4 antibody; and by immunoblotting using this antibody we show these cells express a 130-150 kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.
authors.affiliation: Department of Biomedical Sciences, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA.
authors.forename: Rees
authors.initials: R
authors.lastname: Burt
authors.forename: Bridget M
authors.initials: BM
authors.lastname: Graves
authors.forename: Ming
authors.initials: M
authors.lastname: Gao
authors.forename: Chaunfu
authors.initials: C
authors.lastname: Li
authors.forename: David L
authors.initials: DL
authors.lastname: Williams
authors.forename: Santiago P
authors.initials: SP
authors.lastname: Fregoso
authors.forename: Donald B
authors.initials: DB
authors.lastname: Hoover
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.forename: Gary L
authors.initials: GL
authors.lastname: Wright
authors.forename: Robert
authors.initials: R
authors.lastname: Wondergem
mesh_terms: D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005439:Flufenamic Acid / Q000494:pharmacology*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D051379:Mice; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010616:Phenanthrenes / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000502:physiology; D050053:TRPM Cation Channels / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology
chemical_list: D000893:Anti-Inflammatory Agents; D004791:Enzyme Inhibitors; D010616:Phenanthrenes; D050053:TRPM Cation Channels; C476766:TRPM4 protein, mouse; D005439:Flufenamic Acid; D019284:Thapsigargin; C091375:9-phenanthrol; D002118:Calcium
keywords: HL-1 cardiomyocytes; TRPM4; [Ca(2+)](i)