9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes
- PMID: 23831210
- PMCID: PMC3788596
- DOI: 10.1016/j.ceca.2013.06.003
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.
Keywords: HL-1 cardiomyocytes; TRPM4; [Ca(2+)](i).
Copyright © 2013 Elsevier Ltd. All rights reserved.
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References
-
- Colquhoun D, Neher E, Reuter H, Stevens CF. Inward current channels activated by intracellular Ca in cultured cardiac cells. Nature. 1981;294:752–754. - PubMed
-
- Launay P, Fleig A, Perraud AL, Scharenberg AM, Penner R, Kinet JP. TRPM4 is a Ca2+-activated nonselective cation channel mediating cell membrane depolarization. Cell. 2002;109:397–407. - PubMed
-
- Ullrich ND, Voets T, Prenen J, Vennekens R, Talavera K, Droogmans G, Nilius B. Comparison of functional properties of the Ca2+-activated cation channels TRPM4 and TRPM5 from mice. Cell Calcium. 2005;37:267–278. - PubMed
-
- Nilius B, Prenen J, Droogmans G, Voets T, Vennekens R, Freichel M, Wissenbach U, Flockerzi V. Voltage dependence of the Ca2+-activated cation channel TRPM4. J Biol Chem. 2003;278:30813–30820. - PubMed
-
- Nilius B, Prenen J, Tang J, Wang C, Owsianik G, Janssens A, Voets T, Zhu MX. Regulation of the Ca2+ sensitivity of the nonselective cation channel TRPM4. J Biol Chem. 2005;280:6423–6433. - PubMed
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