Opening of Mitochondrial K _ATP Channels Attenuates the Ouabain-Induced Calcium Overload in Mitochondria

Abstract

2؉] m ) and ⌬⌿ m in rat ventricular myocytes were measured by loading cells with Rhod-2 and JC-1, respectively. Exposure to ouabain (1 mmol/L) for 30 minutes produced mitochondrial Ca 2؉ overload, and the intensity of Rhod-2 fluorescence significantly increased to 173؎16% of baseline (P<0.001). Treatment of myocytes with the mitoK ATP channel opener diazoxide (100 mol/L) blunted the ouabain-induced mitochondrial Ca 2؉ overload (131؎10% of baseline; P<0.001 versus ouabain). Moreover, diazoxide significantly depolarized the ⌬⌿ m and reduced the intensity of JC-1 fluorescence during application of ouabain to 89؎2% of baseline (P<0.05). These effects of diazoxide were blocked by the mitoK ATP channel blocker 5-hydroxydecanoate (500 mol/L). These results indicate that opening of mitoK ATP channels prevents a mitochondrial Ca 2؉ overload in association with ⌬⌿ m depolarization and thereby protects myocardium against ischemic damage. M itochondrial ATP-sensitive Kϩ (mitoK ATP ) channels are thought to play a key role in cardioprotection, 1 but the crucial question remains as to why the opening of mitoK ATP channels can be so protective. Liu et al 2 originally hypothesized that the K ϩ entry through mitoK ATP channels depolarizes the ⌬⌿ m , which reduces the driving force for Materials and Methods Cell PreparationAdult rat ventricular myocytes were isolated by collagenase digestion, as previously described. The Ca 2ϩ fluorophore Rhod-2 was used to measure changes of [Ca 2ϩ ] m . Myocytes were loaded with 10 mol/L Rhod-2 acetoxymethyl ester (Molecular Probes) for 120 minutes at 4°C and then incubated for 30 minutes at 37°C in the culture medium. This two-step cold loading/warm incubation protocol achieves exclusive loading of Rhod-2 into the mitochondria. 6 The ⌬⌿ m was monitored with a fluorescent probe, JC-1 (Molecular Probes). Myocytes were incubated with 0.5 mol/L JC-1 for 10 minutes at 37°C. 7 We verified that the subcellular distribution of fluorescence arising from Rhod-2 and JC-1 was virtually identical to that observed by loading myocytes with the mitochondrial dye rhodamine-123.Myocytes loaded with Rhod-2 and JC-1 were perfused with a HEPES-buffered physiological solution (37°C) containing (mmol/L) NaCl 123, KCl 5, CaCl 2 2.7, MgCl 2 1, HEPES 5, and glucose 5.5 (pH 7.4) and imaged with a Nipkow disk confocal system (CSU10, Yokogawa, Japan), as previously described. 5 Rhod-2 was excited at 488 nm by an argon ion laser, with emission collected above 515 nm through a long-pass barrier filter. JC-1 was excited at 488 nm, and the red emission fluorescence was detected using a long-pass filter of 580 nm. The emission light was imaged through a relay lens to an intensified CCD camera. Images were recorded on a computer (Macintosh 8500/120) at video rate and analyzed with NIH Image 1.62f software. Statistical AnalysisData are expressed as meanϮSEM. Intergroup comparisons are made by Student's t test for two groups and by ANOVA followed by Tukey's test for multiple groups. A value of PϽ0.05 was regarded as significan...

Amiodarone Inhibits Sarcolemmal but Not Mitochondrial KATP Channels in Guinea Pig Ventricular Cells

“…4). The experimental model of ouabain-induced mitochondrial Ca 2ϩ overload was used in our earlier study, in which we showed that in rat cardiomyocytes the mitoK ATP channel opener diazoxide attenuated the mitochondrial Ca 2ϩ overload and such effect associated with the depolarization of mitochondrial membrane potential (Ishida et al, 2001). The present study confirms that diazoxide prevents the mitochondrial Ca 2ϩ overload in guinea pig ventricular myocytes.…”

“…We previously reported that the opening of mitoK ATP channels by diazoxide attenuates the mitochondrial Ca 2ϩ overload in rat ventricular myocytes (Ishida et al, 2001). We therefore examined the effect of amiodarone on mitochondrial Ca 2ϩ overload.…”

Stanniocalcin 1 Prevents Cytosolic Ca2+ Overload and Cell Hypercontracture in Cardiomyocytes

“…21 We have recently revealed that exposure of rat cardiomyocytes to 1 mmol/L ouabain results in mitochondrial Ca 2+ overload and cell hypercontracture. 22,23 Moreover, Liu et al have shown the ouabain-altered interaction of a fraction of the Na + -K + ATPase, leading to the generation of reactive oxygen species from mitochondria, independent of that leading to the increase in [Ca 2+ ]i. 24 Therefore, STC1 may directly or indirectly act on the mitochondria and prevent ouabaininduced cell hypercontracture.…”

Contribution of Sarcoplasmic Reticulum Ca2+ Release and Ca2+ Transporters on Sarcolemmal Channels to Ca2+ Transient in Fetal Mouse Heart

“…Briefly, the hearts were stained by immersion in a 20 M solution of the Ca 2ϩ -sensitive fluorescent indicator Fluo-3 AM (Molecular Probes, Eugene, OR) for 15 min at 37°C. After loading, the heart was transferred to a chamber on a microscope (OLYMPUS IX-70) and then washed out at 37°C with control solution (18) …”