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. 2013 Sep;54(3):193-201.
doi: 10.1016/j.ceca.2013.06.003. Epub 2013 Jul 5.

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

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

Rees Burt et al. Cell Calcium. 2013 Sep.

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.

Keywords: HL-1 cardiomyocytes; TRPM4; [Ca(2+)](i).

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Figures

Fig. 1
Fig. 1
Effect of 9-phenanthrol (10 μM) on [Ca2+]i oscillations recorded by Fura2 fluorescence imaging of HL-1 mouse cardiomyocytes. A. [Ca2+]i versus time in three independently oscillating cells. B. [Ca2+]i versus time. Mean ± SE (n = 40 cells)
Fig. 2
Fig. 2. Sequential effects of 9-phenanthrol (10 μM) and NMDG+ substitution for all but 10 mM external Na+ on [Ca2+]i in a HL-1 cardiomyocyte superfused with Ca2+-free external salt solution
Fig. 3
Fig. 3
Effect of flufenamic acid (10 & 100 μM) on [Ca2+]i oscillations recorded by Fura2 fluorescence imaging of HL-1 mouse cardiomyocytes. A. i & ii Effect of flufenamic acid on [Ca2+]i in single cells; iii. [Ca2+]i versus time. Mean ± SE (n = 40 cells). B. i & ii Effect of flufenamic acid on [Ca2+]i in single cells in Ca2+ -free external solution. iii. [Ca2+]i versus time in Ca2+-free solutiojn. Mean ± SE (n = 35 cells).
Fig. 4
Fig. 4
Sequential effects of store depletion of SR/ER Ca2+ via SERCA pump inhibition by thapsigargin (TG, 10 μM). A. Effect of Ca2+-free solution and 9-phenanthrol (10 μM) on [Ca2+]i in mouse HL-1 cardiomyocytes after store depletion. B. Effect of Ca2+-free solution and NMDG+ substitution on [Ca2+]i in mouse HL-1 cardiomyocytes after store depletion. Mean ± SE (n = 40 cells)
Fig. 5
Fig. 5
Effect of NMDG+ substitution for all but 10 mM of external Na+ on [Ca2+]I in HL-1 cardiomyocytes. A. HL-1 cells superfused with standard external salt solution before and after NMDG+ substitution. Mean ± SE (n = 40 cells). B. HL-1 cells superfused with Ca2+-free external salt solution prior to NMDG+ substitution. Mean ± SE (n = 40 cells)
Fig. 6
Fig. 6
Fluorescent immunohistochemistry labeling of TRPM4 protein with anti-TRPM4 antibody in HL-1 cardiomyocytes. A. Punctate labeling for TRPM4 was associated with the plasma membrane of some HL-1 cells, which were stained without Triton X-100 treatment. B. As in A, but also showing a cell exhibiting higher density punctate labeling. C. As in A & B, but cells permeabilized with TritonX-100. D. Control cells without anit-TPRM4 antibody. Nuclei were counterstained with Hoechst. Scale bar = 25 μm
Fig 7
Fig 7
Immuno-identification of TRPM4 expression in HL-1 cells. Confluent HL-1 cell cultures were homogenized and fractionated by centrifugation. Equal protein samples (100 μg/lane) of each fraction were separated by SDS-PAGE and blotted to PVDF. The blot was reacted with anti-TRPM4 antibody. Lane: 1) protein marker; 2) Whole-cell lysate; 3) Low-speed supernatant (10,000g); 4) Nuclei and mitochondria lysate; 5) High-speed supernatant (45,000g); 6) Membrane protein.
Fig. 8
Fig. 8
Mitochondrial membrane potential (□mito) measured in HL-1 cells using the potentiometric dye TMRM. A. Representative images of HL-1 cells loaded with TMRM and treated with: Control, vehicle control (0.1% DMSO, 9-Phenanthrol (10 μM) or FCCP (1 μM). B Inhibition by 9-phenanthrol (p < 0.05) of TMRM relative fluorescent intensity (RFI) of mitochondrial regions. Mean ± SE (n = 4). C. Inhibition by flufenamic acid (p < 0.05 at 10 & 100 μM) of TMRM relative fluorescent intensity (RFI) of mitochondrial regions. Mean ± SE (n = 4)
Fig. 9
Fig. 9
Effect of 9-phenanthrol (10 μM) on membrane ion channel activity recorded in HL-1 cells from patch-clamp electrophysiology in the on-cell recording mode. A. On-cell recording showing ion channels with predominant inward current. B. On-cell recording showing ion channels with both inward and outward currents. Inset. Displays expanded record of ion channel outward currents. Current and time base applies to A & B.

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