Pharmacological Discrimination of Plasmalemmal and Mitochondrial Sodium–Calcium Exchanger in Cardiomyocyte-Derived H9c2 Cells

Abstract

The Na + -Ca 2+ exchanger (NCX) is involved in the physiological and pathophysiological regulation of intracellular Ca 2+ concentration in various cell types including cardiomyocytes. Although the major role of plasmalemmal NCX in cardiomyocytes is to extrude Ca 2+ from the cell and maintain low cytoplasmic Ca 2+ concentration, 1) NCX is also considered to be responsible for the pathogenesis of ischemia-reperfusion and arrhythmia.2,3) Concerning pharmacological inhibition of NCX, SEA0400 {2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline}, a benzyloxyphenyl compound, was established as a potent inhibitor of NCX with minimum effects on other plasmalemmal ion channels and transporters. [4][5][6] In voltage-clamped guinea pig ventricular myocytes, 1 µM SEA0400, which inhibited the plasmalemmal NCX current by more than 80%, had no effect on the Na + current, L-type Ca 2+ current, delayed rectifier K + current, inwardly rectifying K + current.5) The involvement of plasmalemmal NCX in ouabaininduced arrhythmogenesis, 7) ischemia-reperfusion injury 8,9)

Fluorescence imaging-based analysis of the mitochondrial permeability transition pore opening in cardiomyocyte-derived H9c2 cells

“…Extrusion of mitochondrial Ca 2+ is mediated primarily via mitochondrial Na + -Ca 2+ exchanger, which is activated by Na + gradients between the matrix and the cytosol [19] and is inhibited by diltiazem [21] . We have shown in our pervious study that inhibition of the mitochondrial uniporter inhibited, while mitochondrial Na + -Ca 2+ exchanger inhibition enhanced, the rise in mitochondrial Ca 2+ concentration on elevation of extramitochondrial Ca 2+ concentration [14] . In the present study, the extramitochondrial Ca 2+ -induced decrease in mitochondrial calcein fluorescence was inhibited by ruthenium red and enhanced by diltiazem (Fig.…”

“…Two dimensional images of calcein fluorescence were obtained under excitation at 488 nm. Tetramethylrhodamine ethylester (TMRE) and Rhod 2 fluorescence was imaged under excitation at 514 nm by confocal and epifluorescence microscopy as in our previous studies [8,13,14] . The time course of the whole cell fluorescence intensity was calculated with Aquacosmos software (Hamamatsu Photonics).…”

Fluorescence Analysis of the Mitochondrial Effect of a Plasmalemmal Na⁺/Ca²⁺ Exchanger Inhibitor, SEA0400, in Permeabilized H9c2 Cardiomyocytes

“…3). SEA0400, at a concentration of 1 µM which shows marked inhibition of the plasmalemmal NCX in various cells including H9c2 cells, 18) had no effect on mitochondrial Ca 2+ both under normal and ischemic conditions. This indicates that SEA0400 inhibits neither the Ca 2+ uniporter nor the mitochondrial NCX, and has no direct effect on mitochondrial Ca 2+ transport pathways.…”

KB-R7943 Inhibits the Mitochondrial Ca²⁺ Uniporter but Not Na⁺–Ca²⁺ Exchanger in Cardiomyocyte-Derived H9c2 Cells

“…The experimental system was basically the same as that described in our previous reports . 7,8) Rat embryonic heart derived H9c2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS. To obtain mitochondrial-targeted yellow cameleon DNA, its full-length cDNA 11) was inserted into an expression vector which has a mitochondria targeting signal sequence, pEYFP-mito (Clontech, Palo Alto, CA, USA).…”

“…Using this system, we previously reported that SEA0400 affects neither mitochondrial Ca 2+ entry nor Ca 2+ extrusion. 7,8) Concerning the effect of KB-R7943 on mitochondrial Ca 2+ transporters, conflicting results have been reported depending on the cell type. 9,10) In the present study, we intended to clarify the effect of KB-R7943 on mitochondrial Ca 2+ transport in cardiomyocytes using the H9c2 cell system described above.…”