9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes

• Published in: Cell calcium (Edinburgh) 2013-09, Vol.54 (3), p.193-201
• Main Authors: Burt, Rees, Graves, Bridget M, Gao, Ming, Li, Chaunfu, Williams, David L, Fregoso, Santiago P, Hoover, Donald B, Li, Ying, Wright, Gary L, Wondergem, Robert
• Format: Article
• Language: English
• Subjects: [Ca2+]i; Advanced Basic Science; Animals; Anti-Inflammatory Agents - pharmacology; Calcium - metabolism; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - physiology; Enzyme Inhibitors - pharmacology; Flufenamic Acid - pharmacology; HL-1 cardiomyocytes; Membrane Potential, Mitochondrial - drug effects; Mice; Myocytes, Cardiac - drug effects; Myocytes, Cardiac - metabolism; Patch-Clamp Techniques; Phenanthrenes - pharmacology; Sarcoplasmic Reticulum - drug effects; Sarcoplasmic Reticulum - physiology; Thapsigargin - pharmacology; TRPM Cation Channels - metabolism; TRPM4
• ISSN: 0143-4160; 1532-1991
• DOI: 10.1016/j.ceca.2013.06.003

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 Ca2+ -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.