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 & 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 kD 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.

The TRPM4 channel inhibitor 9‐phenanthrol

“…The results summarized earlier indicate that 9‐phenanthrol acts in sinoatrial node cells to directly slow the diastolic depolarization through inhibiton of TRPM4 channels. However, a study conducted on the HL‐1 cell line derived from mouse atrial cells revealed that 9‐phenanthrol also modulates cytoplasmic calcium oscillations (Burt et al ., ). HL‐1 cells are derived from atrial cardiomyocytes, but not specifically sinoatrial node cells, and they acquire properties similar to those of pacemaker cells and develop free beating activity, with intracellular calcium oscillations being present in about 40% of the cells (Wondergem et al ., ).…”

“…HL‐1 cells are derived from atrial cardiomyocytes, but not specifically sinoatrial node cells, and they acquire properties similar to those of pacemaker cells and develop free beating activity, with intracellular calcium oscillations being present in about 40% of the cells (Wondergem et al ., ). Fluorescent Ca 2+ recordings using Fura‐2 revealed that 10 −5 mol·L −1 9‐phenanthrol abolishes these Ca 2+ oscillations leading to a transient increase in cytoplasmic Ca 2+ , attributed to the release of Ca 2+ from an intracellular pool different from the sarcoplasmic reticulum and thus, is most likely to be mitochondrial Ca 2+ (Burt et al ., ). These Ca 2+ oscillations could participate in the spontaneous activity, since the application of 10 −5 mol·L −1 9‐phenanthrol in cell‐attached recordings from HL‐1 cells abolished the depolarizing inward ionic currents that contribute to the slow diastolic depolarization (Burt et al ., ).…”

“…Thirdly, 9‐phenanthrol autofluoresces under UV light (Moriconi et al ., ) in particular at 340 nm, which can induce artefacts when measuring Ca 2+ or voltage using fluorescent dyes (Burt et al ., ). This can cause problems when using high concentrations.…”

“…(iii) The IC 50 values for the effects of 9‐phenanthrol on physiological processes are in the same range as those measured for TRPM4 channel inhibition in recombinant HEK cells (Tables ). (iv) The effect of 9‐phenanthrol can be reproduced by other TRPM4 inhibitors such as flufenamic acid (Gonzales et al ., 2010a; Saito and Yanagawa, ; Mrejeru et al ., ; Shpak et al ., ; Simard et al ., 2012a; 2013; Amarouch et al ., ; Burt et al ., ; Kim et al ., ) and glibenclamide (Becerra et al ., ; Kim et al ., ).…”

The transient receptor potential melastatin 4 channel inhibitor 9‐phenanthrol modulates cardiac sodium channel

“…9‐Phenanthrol (10–100 μM) has also been reported to mitigate calcium oscillation in HL‐1 mouse cardiomyocytes (Burt et al, ) and terminate burst firing in a neuronal system (Mrejeru et al, ), and again these may partially attributed to sodium current inhibition.…”