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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...

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