Opening of mitochondrial K(ATP) channels attenuates the ouabain-induced calcium overload in mitochondria.

We tested whether opening of mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channels depolarizes mitochondrial membrane potential (DeltaPsi(m)) and thereby prevents the mitochondrial Ca(2+) overload. With the use of a Nipkow disk confocal system, the mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and DeltaPsi(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 micromol/L) blunted the ouabain-induced mitochondrial Ca(2+) overload (131+/-10% of baseline; P<0.001 versus ouabain). Moreover, diazoxide significantly depolarized the DeltaPsi(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 micromol/L). These results indicate that opening of mitoK(ATP) channels prevents a mitochondrial Ca(2+) overload in association with DeltaPsi(m) depolarization and thereby protects myocardium against ischemic damage.

Physiological concentration of nitric oxide induces positive inotropic effects through cGMP pathway in isolated rat ventricular myocytes.

We used authentic NO or NO from NO donors to show that the physiological levels of NO (<1 microM) induce a positive inotropic effect and demonstrated that the effect is evoked through a cGMP-dependent pathway. In isolated rat ventricular myocytes, authentic NO at 588 nM increased both cell shortening and the intracellular Ca(2+) ([Ca(2+)]i) transient (133 and 117%, respectively; p < 0.05 vs. baseline), and 0.16-1.7 microM NO elicited reproducible dose-dependent increases in cell shortening. NOC18 (0.1 mM: actual NO concentration 673 nM) or SNAP (0.1 mM: actual NO concentration 285 nM) showed similar effects (shortening 215% and [Ca(2+)]i transient 160% increases, and shortening 148% and [Ca(2+)]i transient 117% increases, respectively). The NO-induced increases in cell shortening and the [Ca(2+)]i transient were inhibited by an inhibitor of soluble guanylate cyclase (ODQ, 30 microM) or by an inhibitor of cAMP-dependent protein kinase (KT5720, 0.1 microM). In the presence of an inhibitor of cGMP-inhibited cAMP-phosphodiesterase (milrinone, 10 microM), NO failed to increase both cell shortening and the [Ca(2+)]i transient. These results suggest that physiological levels of NO induce positive inotropy through a cGMP-dependent pathway.

Haloperidol prolongs diastolic phase of Ca(2+) transient in cardiac myocytes.

Haloperidol (HPL), a widely used antipsychotic drug, is known to induce serious ventricular arrhythmias. However, the mechanism underlying their induction is not clear. We therefore examined the effects of HPL on the intracellular Ca(2+) ([Ca(2+)](i)) transient and on cell motion in cultured cardiac myocytes, as well as the pathways involving the HPL-induced abnormality of Ca(2+) homeostasis. HPL prolonged the diastolic phase of the Ca(2+) transient, with a mid-diastolic re-elevation of [Ca(2+)](i). The re-elevation of [Ca(2+)](i) was shown to be provoked by Ca(2+) release from sarcoplasmic reticulum (SR), which can trigger delayed afterdepolarization, the major arrhythmogenic factor. The re-elevation of [Ca(2+)](i) coincided with cell re-contraction during diastole. The induction of this abnormality by HPL appears to be independent of the mechanisms of the antipsychotic action.

Formation of planar and spiral Ca2+ waves in isolated cardiac myocytes.

A novel Nipkow-type confocal microscope was applied to image spontaneously propagating Ca2+ waves in isolated rat ventricular myocytes by means of fluo-3. The sarcolemma was imaged with di-8-ANEPPS and the nucleus with SYTO 11. Full frame images in different vertical sections were obtained at video frame rate by means of an intensified CCD camera. Three types of Ca2+ waves were identified: spherical waves, planar waves, and spiral waves. Both spherical waves and spiral waves could initiate a planar wave, and planar waves were not influenced by the presence of a nucleus. Spiral waves, however, were consistently found adjacent to a nucleus and displayed a slower propagation rate and slower rate of increase in Ca2+ concentration in the wave front than did spherical and planar waves. The planar waves were apparent throughout the vertical axis of the cell, whereas spiral waves appeared to have a vertical height of approximately 3 microm, less than the maximum thickness of the nucleus (5.0 +/- 0.3 microm). These results provide experimental confirmation of previous modeling studies which predicted an influence of the nucleus on spiral-type Ca2+ waves. When a spontaneous Ca2+ wave is small relative to the size of the nucleus, it appears that the Ca2+ buffering by the nucleus is sufficient to slow the rate of spontaneous propagation of the Ca2+ wave in close proximity to the nucleus. These findings thus support the idea that the nucleus can influence complex behavior of Ca2+ waves in isolated cardiac myocytes.

Distinct roles of peroxynitrite and hydroxyl radical in triggering stunned myocardium-like impairment of cardiac myocytes in vitro.

Myocardial stunning is characterized by the impairment of excitation-contraction coupling via a decrease in myofilament Ca2+ responsiveness, thought to be triggered by hydroxyl radicals (*OH) generated upon reperfusion. Since peroxynitrite is also expected to be produced during reperfusion, we examined whether it can induce a stunned myocardium-like impairment of cardiac myocytes. Its effect on cultured cardiac myocytes was compared with that of hydrogen peroxide (H2O2), *OH source. Infusion of peroxynitrite (0.2 mM) induced a decrease in cell motion and a complete arrest in diastole at 2.9 +/- 0.3 min, which coincided with an elevation in [Ca2+]i. Arrest induced by infusion of H2O2 (10 mM) was not associated with an increase in [Ca2+]i. The ATP content was unaffected by peroxynitrite (control, 34.3 +/- 3.4: + peroxynitrite, 32.9 +/- 3.5 nmol/mg protein) and the cells remained viable. Sulfhydryl (SH) content was decreased by peroxynitrite, but not by H2O2. The membrane fluidity (a measure of peroxidation of the membrane lipids) was not affected by peroxynitrite, but was decreased by H2O2. Onset time of arrest was unaffected by deferoxamine (0.2 mM), but was delayed by DTT (10 mM) (from 2.9 +/- 0.3 to 19.2 +/- 1.6 min). Nitrotyrosine content was unchanged by peroxynitrite, and its augmentation with Fe3+/EDTA (1 mM) was not associated with a shortened onset time of arrest. The function of the Na+/Ca2+ exchanger was impaired by peroxynitrite, but not by H2O2. Peroxynitrite and H2O2 each induce arrest, but only the former increases [Ca2+]i. One of the mechanisms of the increase in [Ca2+]i is Na/Ca2+ exchanger dysfunction. The impairments were induced through SH oxidation by peroxynitrite, but through lipid peroxidation by H2O2. Myocardial stunning may be induced by both species in concert.

Visualization of biphasic Ca2+ diffusion from cytosol to nucleus in contracting adult rat cardiac myocytes with an ultra-fast confocal imaging system.

In contracting cardiac myocytes, the rapid changes in cytosolic and nuclear Ca2+ make it difficult to determine whether the nuclear Ca2+ transient is caused by diffusion from the cytosol or by Ca2+ release channels on the inner nuclear membrane, or both. The propagation mechanism in the nucleoplasm also remains unknown. We have developed an ultra-fast Nipkow confocal imaging system able to acquire two-dimensional images at approximately 4 ms/full frame speed and employed it to analyze Ca2+ waves and the dynamics of the cytosolic and nuclear Ca2+ transients after electrical stimulation of cardiac myocytes. The pattern of nuclear Ca2+ upon stimulation was well described by a mathematical model of Ca2+ diffusion across the nuclear envelope. No evidence of Ca2+ release from perinuclear Ca2+ stores was obtained. The Ca2+ diffusion constant appeared to change during contraction, with essentially free diffusion of Ca2+ through nuclear pore complexes at low cytosolic Ca2+ and partially restricted diffusion at high cytosolic Ca2+. The Ca2+ in the nucleoplasm propagated by diffusion and no Ca2+ release phenomena were seen in the nucleus.

Application of authentic peroxynitrite to biological materials.

A method to expose cultured cardiac myocytes to authentic ONOO- by using a constant perfusion system was described. The perfusion apparatus is linked to a microscope equipped with video and fluorescence measurement systems, which enable the evaluation of myocardial contraction, [Ca2+]i, [pH]i, and membrane fluidity. Special care is needed to adjust the pH of the solution appropriately and to obtain a constant concentration of ONOO-.

Peroxynitrite is not a major mediator of endothelial cell injury by activated neutrophils in vitro.

OBJECTIVE: Human polymorphonuclear leukocytes (PMN) produce nitric oxide (NO), superoxide (O2.-) and peroxynitrite (ONOO-) upon stimulation. We investigated the role of ONOO- in PMN-induced injury to cultured bovine aortic endothelial cells (BAEC). METHODS: BAEC were cocultured with phorbol 12-myristate 13-acetate (PMA)-activated human PMN (effector-to-target ratio, 10:1) and injury to BAEC was evaluated at intervals by 51Cr release assay. The levels of NO, O2.-, ONOO- and nitrotyrosine, a reaction product of ONOO-, were also measured, and the influence of NO synthase inhibitors, O2.- and hydroxyl radical scavengers and other effectors was examined. RESULTS: In BAEC cocultured with PMA-activated PMN, 51Cr release was significantly increased [14.6 +/- 2.2% at 2 h (p < 0.05) and 42.6 +/- 2.7% at 4 h (p < 0.01); control (nonactivated PMN), < 4%]. Superoxide dismutase (100 U/ml) reduced 51Cr release to 4.6 +/- 2.2% at 2 h (p < 0.05). N-Iminoethyl-L-ornithine (L-NIO, 0.1 mM) potentiated 51Cr release (30.6 +/- 3.8% at 2 h, p < 0.01), and the potentiation was eliminated by anti-CD18 monoclonal antibody. The 51Cr release was completely prevented by dimethyl sulfoxide or by deferoxamine. Treatment of PMN with L-NIO inhibited NO generation and increased O2.- production. The nitrotyrosine level did not increase in BAEC cocultured with PMA-activated PMN. CONCLUSION: NO-derived ONOO- is not a major cytotoxic mediator in BAEC injury by activated PMN. NO may have a cytoprotective effect by inhibiting PMN adherence to endothelial cells.