9-Phenanthrol and flufenamic acid inhibit calcium oscillations in HL-1 mouse cardiomyocytes
ABSTRACT It is well established that intracellular calcium ([Ca(2+)]i) controls the inotropic state of the myocardium, and evidence mounts that a "Ca(2+) 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 Ca(2+) 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 (10μM) and flufenamic acid (10 and 100μM) decreases Ca(2+) oscillations followed by an overall increase in [Ca(2+)]i. The latter occurs also in HL-1 cells in Ca(2+)-free solution and after depletion of sarcoplasmic reticulum Ca(2+) with thapsigargin (10μM). 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 130-150kDa protein, as expected for TRPM4. We conclude that 9-phenanthrol inhibits TRPM4 ion channels in HL-1 cells, which in turn decreases Ca(2+) oscillations followed by a compensatory increase in [Ca(2+)]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.
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ABSTRACT: TRPM4 is a non-selective Ca2+-activated cation channel expressed in the heart, particularly in the atria or conduction tissue. Mutations in the Trpm4 gene were recently associated with several human conduction disorders such as Brugada syndrome. TRPM4 channel has also been implicated at the ventricular level, in inotropism or in arrhythmia genesis due to stresses such as ß-adrenergic stimulation, ischemia-reperfusion, and hypoxia re-oxygenation. However, the physiological role of the TRPM4 channel in the healthy heart remains unclear. We aimed to investigate the role of the TRPM4 channel on whole cardiac function with a Trpm4 gene knock-out mouse (Trpm4-/-) model. Morpho-functional analysis revealed left ventricular (LV) eccentric hypertrophy in Trpm4-/- mice, with an increase in both wall thickness and chamber size in the adult mouse (aged 32 weeks) when compared to Trpm4+/+ littermate controls. Immunofluorescence on frozen heart cryosections and qPCR analysis showed no fibrosis or cellular hypertrophy. Instead, cardiomyocytes in Trpm4-/- mice were smaller than Trpm4+/+with a higher density. Immunofluorescent labeling for phospho-histone H3, a mitosis marker, showed that the number of mitotic myocytes was increased 3-fold in the Trpm4-/-neonatal stage, suggesting hyperplasia. Adult Trpm4-/- mice presented multilevel conduction blocks, as attested by PR and QRS lengthening in surface ECGs and confirmed by intracardiac exploration. Trpm4-/-mice also exhibited Luciani-Wenckebach atrioventricular blocks, which were reduced following atropine infusion, suggesting paroxysmal parasympathetic overdrive. In addition, Trpm4-/- mice exhibited shorter action potentials in atrial cells. This shortening was unrelated to modifications of the voltage-gated Ca2+ or K+ currents involved in the repolarizing phase. TRPM4 has pleiotropic roles in the heart, including the regulation of conduction and cellular electrical activity which impact heart development.PLoS ONE 12/2014; 9(12):e115256. DOI:10.1371/journal.pone.0115256 · 3.53 Impact Factor
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ABSTRACT: The phenanthrene-derivative 9-phenanthrol is a recently identified inhibitor of the TRPM4 Ca(2+) -activated non-selective cation channel whose mechanism of action remains to be determined. Subsequent studies performed on other ion channels confirm the specificity of the drug for TRPM4. In addition, 9-phenanthrol modulates a variety of physiological processes through TRPM4 current inhibition and thus exerts beneficial effects in several pathological conditions. 9-phenanthrol modulates smooth muscle contraction in bladder and cerebral arteries, impacts spontaneous activity in neurons and heart, and reduces lipopolysaccharide-induced cell death. Among promising potential applications, 9-phenanthrol exerts cardioprotective effects against ischemia-reperfusion injuries and reduces ischemic stroke injuries. In addition to reviewing the biophysical effects of 9-phenanthrol, here we present information about its appropriate use in physiological studies and possible clinical applications.British Journal of Pharmacology 01/2014; DOI:10.1111/bph.12582 · 4.99 Impact Factor
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ABSTRACT: The non-selective Transient Receptor Potential Melastatin 4 (TRPM4) cation channel is abundantly expressed in cardiac cells, being involved in several aspects of cardiac rhythmicity, including cardiac conduction, pace making and action-potential repolarization. Dominantly inherited mutations in the TRPM4 gene are associated with the cardiac bundle-branch disorder progressive familial heart block type I (PFHBI) and isolated cardiac conduction disease (ICCD) giving rise to atrio-ventricular conduction block (AVB), right bundle branch block, bradycardia, and the Brugada syndrome. The mutant phenotypes closely resemble those associated with mutations in the SCN5A gene, encoding the voltage-gated Na(+) channel NaV1.5. These observations and the unexpected partnership with sulfonylurea-receptors (SURs) makes the TRPM4 channel a promising novel target for treatment of cardiac disorders.Current Opinion in Pharmacology 04/2014; 15C:68-73. DOI:10.1016/j.coph.2013.12.003 · 4.23 Impact Factor