Masatsugu Hori

Delayed effects of sublethal ischemia on the acquisition of tolerance to ischemia.

The infarct-limiting effect of ischemic preconditioning is believed to be a transient phenomenon. We examined the delayed effects of repetitive brief ischemia on limiting infarct size in an open-chest dog model by an occlusion (90 minutes) of the left anterior descending coronary artery (LAD) followed by reperfusion (5 hours). The dogs were preconditioned with four brief repeated ischemic episodes induced by 5-minute LAD occlusions with subsequent reperfusion. The size of infarcts initiated by a sustained occlusion immediately or 24 hours after preconditioning was significantly smaller when compared with infarcts in sham-operated dogs (for the immediate occlusion, 14.4 +/- 2.0% versus 39.0 +/- 3.7%, respectively [p < 0.01]; and for the delayed occlusion, 18.8 +/- 3.4% versus 35.1 +/- 4.6%, respectively [p < 0.05]); however, when the infarction was induced 3 hours (31.2 +/- 3.7% versus 37.5 +/- 4.2%, respectively) or 12 hours (25.4 +/- 4.8% versus 35.0 +/- 5.3%, respectively) after repetitive ischemia, the infarct size did not differ. No differences were seen in regional myocardial blood flow or rate-pressure products between the two groups. These results indicate that an infarct-limiting effect of brief repeated ischemia can be observed 24 hours after sublethal preconditioning.

Inactivation of Glutathione Peroxidase by Nitric Oxide IMPLICATION FOR CYTOTOXICITY

S-nitro-N-acetyl-DL-penicillamine (SNAP), a nitric oxide (NO) donor, inactivated bovine glutathione peroxidase (GPx) in a dose- and time-dependent manner. The IC of SNAP for GPx was 2 μM at 1 h of incubation and was 20% of the IC for another thiol enzyme, glyceraldehyde-3-phosphate dehydrogenase, in which a specific cysteine residue is known to be nitrosylated. Incubation of the inactivated GPx with 5 mM dithiothreitol within 1 h restored about 50% of activity of the start of the SNAP incubation. A longer exposure to NO donors, however, irreversibly inactivated the enzyme. The similarity of the inactivation with SNAP and reactivation with dithiothreitol of GPx to that of glyceraldehyde-3-phosphate dehydrogenase, suggested that NO released from SNAP modified a cysteine-like essential residue on GPx. When U937 cells were incubated with 100 μM SNAP for 1 h, a significant decrease in GPx activity was observed although the change was less dramatic than that with the purified enzyme, and intracellular peroxide levels increased as judged by flow cytometric analysis using a peroxide-sensitive dye. Other major antioxidative enzymes, copper/zinc superoxide dismutase, manganese superoxide dismutase, and catalase, were not affected by SNAP, which suggested that the increased accumulation of peroxides in SNAP-treated cells was due to inhibition of GPx activity by NO. Moreover, stimulation with lipopolysaccharide significantly decreased intracellular GPx activity in RAW 264.7 cells, and this effect was blocked by NO synthase inhibitor N-methyl-L-arginine. This indicated that GPx was also inactivated by endogenous NO. This mechanism may at least in part explain the cytotoxic effects of NO on cells and NO-induced apoptotic cell death.

Induction of manganese superoxide dismutase in rat cardiac myocytes increases tolerance to hypoxia 24 hours after preconditioning.

Manganese superoxide dismutase (Mn-SOD) is induced in ischemic hearts 24 h after ischemic preconditioning, when tolerance to ischemia is acquired. We examined the relationship between Mn-SOD induction and the protective effect of preconditioning using cultured rat cardiac myocytes. Exposure of cardiac myocytes to brief hypoxia (1 h) decreased creatine kinase release induced by sustained hypoxia (3 h) that follows when the sustained hypoxia was applied 24 h after hypoxic preconditioning (57% of that in cells without preconditioning). The activity and content of Mn-SOD in cardiac myocytes were increased 24 h after hypoxic preconditioning (activity, 170%; content, 139% compared with cells without preconditioning) coincidentally with the acquisition of tolerance to hypoxia. Mn-SOD mRNA was also increased 20-40 min after preconditioning. Antisense oligodeoxyribonucleotides corresponding to the initiation site of Mn-SOD translation inhibited the increases in the Mn-SOD content and activity and abolished the expected decrease in creatine kinase release induced by sustained hypoxia after 24 h of hypoxic preconditioning. Sense oligodeoxyribonucleotides did not abolish either Mn-SOD induction or tolerance to hypoxia. These results suggest that the induction of Mn-SOD in myocytes by preconditioning plays a pivotal role in the acquisition of tolerance to ischemia at a later phase (24 h) of ischemic preconditioning.

Effect of angina pectoris on myocardial protection in patients with reperfused anterior wall myocardial infarction: retrospective clinical evidence of "preconditioning".

OBJECTIVESnWe examined whether angina pectoris occurring shortly before the onset of acute myocardial infarction can actually preserve postischemic left ventricular function in humans.nnnBACKGROUNDnExperimental studies indicate that brief, transient episodes of ischemia render the heart very resistant to infarction from a subsequent sustained ischemic insult, an effect termed ischemic preconditioning. However, no clinical data are available concerning the implications of angina pectoris shortly before the onset of infarction in humans.nnnMETHODSnWe studied 84 patients with an acute anterior myocardial infarction. All patients had total occlusion of the proximal or medial portion of the left anterior descending coronary artery and achieved reflow within 6 h of onset. Patients were classified into three groups on the basis of duration of antecedent angina pectoris: group 1 = no angina (37 patients); group 2 = new angina pectoris occurring < or = 7 days of onset of infarction (22 patients); group 3 = angina pectoris beginning > 7 days before onset of infarction (25 patients). All patients underwent left ventriculography on the day of, and 28 days after, onset of infarction to determine ejection fraction and regional wall motion in the territory of the left anterior descending coronary artery by the centerline method.nnnRESULTSnAngiographic collateral flow grade was higher in group 3 than in groups 1 and 2 ([mean +/- SD] group 1 = 0.08 +/- 0.7, group 2 = 0.7 +/- 0.7, group 3 = 1.5 +/- 0.8). Although there were no differences in baseline ejection fraction and regional wall motion among the three groups, the degree of improvement was significantly greater in groups 2 and 3 than in group 1 (late minus baseline ejection fraction: group 1 = 0 +/- 8%, group 2 = 7 +/- 10% group 3 = 6 +/- 10% [p < 0.05 group 1 vs. groups 2 and 3]; late minus baseline regional wall motion: group 1 = 0.2 +/- 0.4, group 2 = 0.6 +/- 0.5, group 3 = 0.5 +/- 0.6 SD/chord [p < 0.05, group 1 vs. group 2]). When the study was limited to those patients with no or poor collateral flow (31 in group 1, 19 in group 2, 10 in group 3), only group 2 patients had a significant improvement in wall motion. Angina pectoris within 24 h before onset of infarction was more frequent in group 2 (82%) than group 3 (28%, p < 0.05).nnnCONCLUSIONSnEpisodes of angina pectoris occurring shortly before the onset of infarction may preserve myocardial contractile function in reperfused myocardial infarction despite less support from collateral flow channels, although these are suggestive results in a limited number of patients.

High prevalence of atrial fibrosis in patients with dilated cardiomyopathy

OBJECTIVESnWe examined the extent of fibrotic changes in the left atrium of cardiomyopathic human hearts and investigated the relation of mechanical overload caused by left ventricular dysfunction to fibrosis of the left atrium.nnnBACKGROUNDnLeft atrial dysfunction in dilated cardiomyopathy may contribute to progression of heart failure. In contrast to fibrosis of the left ventricle, atrial fibrosis has not been extensively studied in cardiomyopathic hearts.nnnMETHODSnThe extent of fibrosis in the left atrium and left ventricle was determined by an automatic image analyzer in 38 autopsied hearts obtained from 9 patients who died of noncardiac illness (control group), 16 patients with dilated cardiomyopathy, 6 patients with hypertrophic cardiomyopathy with features mimicking dilated cardiomyopathy and 7 patients with a previous myocardial infarction. Transverse sections were obtained at the upper margins of the foramen ovale and left auricle in the left atrium and the median level of the left ventricle.nnnRESULTSnThere were no significant differences in extent of left atrial dilation, left ventricular dysfunction or duration of illness among the three groups with cardiac disease. Percent area of left atrial fibrosis (mean +/- SD) was significantly greater in the specimens from patients with dilated cardiomyopathy (13.1 +/- 6.1%, p < 0.01) and hypertrophic cardiomyopathy mimicking dilated cardiomyopathy (26.5 +/- 9.5%, p < 0.01) than in those from patients with an old myocardial infarction (3.8 +/- 1.1%). Percent area of left ventricular fibrosis in hearts from patients with dilated cardiomyopathy (12.9 +/- 8.6%) was significantly smaller than that in hearts from patients with hypertrophic cardiomyopathy mimicking dilated cardiomyopathy (35.8 +/- 11.9%, p < 0.01) and a previous myocardial infarction (38.4 +/- 8.0%, p < 0.01). Percent area of atrial fibrosis was significantly correlated with left ventricular ejection fraction in the group with a previous myocardial infarction but not in the other groups.nnnCONCLUSIONSnThere was a high degree of fibrotic change in the left atrium in the groups with dilated cardiomyopathy and hypertrophic cardiomyopathy mimicking dilated cardiomyopathy. Our findings suggest that atrial fibrosis in these patients may not have been related to mechanical overload of the left atrium but to some other, still unknown mechanisms.

Amelioration of severity of myocardial injury by a nitric oxide donor in rabbits fed a cholesterol-rich diet☆

OBJECTIVESnThis study compared the effect of a nitric oxide donor on limiting the size of infarct resulting from myocardial ischemia-reperfusion between atherosclerotic and nonatherosclerotic models.nnnBACKGROUNDnEndothelial-derived relaxation in coronary arteries affected by ischemia is substantially impaired after reperfusion, and this impairment may exacerbate the myocardial ischemia-reperfusion injury. In animals with experimental atherosclerosis, release of endothelial-derived relaxing factor is also decreased, and the propagation of myocardial infarction could be exacerbated.nnnMETHODSnWe examined the extent of myocardial injury induced by ischemia (30 min) and reperfusion (48 hr) in rabbits fed a cholesterol-rich (1%) or normal diet for 10 weeks. We also evaluated the effect of a nitric oxide donor (S-nitroso-N-acetylpenicillamine [SNAP], a nitric oxide precursor (L-arginine) or a degradation product of SNAP (N-acetylpenicillamine) on infarct size in these models.nnnRESULTSnSeverity of myocardial injury was significantly exacerbated in cholesterol-fed rabbits (75.2 +/- 4.4% [mean +/- SEM]) compared with that in non-cholesterol-fed rabbits (53.2 +/- 5.2%). This exacerbation was prevented by treatment with SNAP (50.2 +/- 6.4%) but not with L-arginine (70.5 +/- 6.0%) or N-acetylpenicillamine (70.4 +/- 4.8%) in cholesterol-fed-rabbits. However, SNAP did not limit infarct size in non-cholesterol-fed rabbits (60.8 +/- 4.2%). The rate-pressure product was similar during the course of the experiment in all the groups.nnnCONCLUSIONSnMyocardial damage induced by ischemia-reperfusion was significantly exacerbated in rabbits fed a long-term cholesterol-rich diet but was effectively reversed by treatment with a nitric oxide donor. However, this agent did not limit infarct size in normal rabbits. Thus, a nitric oxide donor reduces myocardial infarct size in atherosclerotic but not in nonatherosclerotic rabbits.

Gamma-glutamylcysteine ethyl ester for myocardial protection in dogs during ischemia and reperfusion

OBJECTIVESnThe aim of this study was to examine the infarct-limiting effects of gamma-glutamylcysteine ethyl ester, a newly discovered synthetic precursor of glutathione biosynthesis, in a canine model of myocardial infarction.nnnBACKGROUNDnReduced glutathione plays an important role in protecting cells against damage induced by reactive oxygen species during myocardial ischemia and reperfusion. Gamma-glutamylcysteine ethyl ester is capable of penetrating into cells in its intact form and increasing intracellular glutathione levels.nnnMETHODSnDogs were subjected to a 90-min coronary occlusion followed by 5 h of reperfusion. An intravenous bolus injection of gamma-glutamylcysteine ethyl ester (3 or 10 mg/kg body weight) was administered immediately before reperfusion. Regional myocardial blood flow was measured with the use of colored microspheres.nnnRESULTSnGamma-glutamylcysteine ethyl ester effectively reduced infarct size in a dose-dependent manner (mean +/- SEM 26.4 +/- 3.5% in the low dose group [3 mg/kg, n = 10] and 19.0 +/- 3.4% in the high dose group [10 mg/kg, n = 10]; each p < 0.05 vs. the value in the control group [40.6 +/- 4.8%, n = 10]). There were no differences between the control and treated groups in hemodynamic variables or regional myocardial blood flow either during the ischemic period or after reperfusion. The reduced glutathione content of ischemic myocardium in the control group (0.62 +/- 0.11 mumol/g, p < 0.01) was significantly lower than that in nonischemic myocardium (1.46 +/- 0.07 mumol/g), and it was preserved by treatment in a dose-dependent manner (3 mg/kg, 0.83 +/- 0.06 mumol/g; 10 mg/kg, 0.92 +/- 0.14 mumol/g; each p < 0.05 vs. control level). There were no differences in oxidized glutathione content between nonischemic and ischemic myocardium or among the three groups.nnnCONCLUSIONSnGamma-glutamylcysteine ethyl ester, a precursor of glutathione, significantly attenuates myocardial ischemia and reperfusion injury when administered immediately before reperfusion.

Brief myocardial ischemia affects free radical generating and scavenging systems in dogs

SummaryThis study examined whether brief repeated myocardial ischemia altered free radical generating and scavenging activity in a dog model. In dogs preconditioned with four 5-min left anterior descending coronary artery (LAD) occlusions and reperfusions, we examined transcardiac changes in both the function of neutrophils, cells which are major free radical generators, and in myocardial antioxidant enzyme activity, as an indication of free radical scavenging. Neutrophil function was assessed by determining luminol-enhanced whole blood chemiluminescence (CL) induced by zymosan. Blood was taken simultaneously from the carotid artery and the cardiac vein running along the occluded LAD. Preconditioning with sublethal ischemia significantly reduced whole blood CL in the cardiac vein compared with the carotid artery after the first and fourth 5-min reperfusions, while there was no difference in neutrophil count between these sampling sites. Immediately after brief repeated ischemia and reperfusion, manganese-superoxide dismutase (SOD) activity was significantly enhanced, and glutathione reductase activity was markedly reduced in the ischemic, compared with the non-ischemic, myocardium. There were no differences in the myocardial activities of copper, zinc-SOD, glutathione peroxidase, and glutathione S-transferase between the ischemic and non-ischemic regions. Also, no difference was observed between the reduced myocardial glutathione levels in these regions, although the oxidized glutathione level was significantly higher in the ischemic regions of the subepicardial and subendocardial areas. We demonstrated that brief repeated ischemia affects free radical generating and scavenging systems in the ischemic myocardium.

Polymorphonuclear leukocytes-induced injury in hypoxic cardiac myocytes

Growing evidence suggests that free radicals derived from polymorphonuclear leukocytes (PMNs) play an important role in myocardial ischemia-reperfusion injury. To elucidate the cellular mechanism by which activated PMNs exacerbate ischemic myocardial damage, we investigated the extent of cell injury, assessed by the morphological deterioration, free radical generation, and lipid peroxidation in mouse embryo myocardial cells coincubated with activated PMNs. The generation of PMN-derived free radicals was related to the extent of myocardial cell injury. When myocardial cell sheets were subjected to hypoxia and glucose-free media, myocardial cells were injured (cristalysis in the mitochondria and disruption of the sarcolemma) after adding various PMN activators, and the injury extended to the adjacent cells. Chemiluminescent emission and production of thiobarbituric acid-reactive substances in the coincubated cells increased markedly compared with myocardial cells or PMNs alone. The augmented lipid peroxidation coincided with the progression of myocardial cell injury. Catalase inhibited the myocardial cell injury by 52%, the chemiluminescence by 46%, and lipid peroxidation by 50%, whereas superoxide dismutase exhibited less pronounced inhibition. These results indicate that a chain reaction of lipid peroxidation in myocardial cells induced by PMN-derived free radicals closely correlates with membrane damage and contributes to the propagation of irreversible myocardial cell damage.

PARTIAL PURIFICATION AND CHARACTERIZATION OF A PROTEIN KINASE THAT IS ACTIVATED BY NUCLEAR LOCALIZATION SIGNAL PEPTIDES

A nuclear localization signal (NLS) is required for the transport of karyophilic proteins from the cytoplasm to the nucleus. In this study, NLS was examined in terms of its effect on diverse cellular functions such as protein phosphorylation reactions. When synthetic peptides containing the NLS of SV40 T‐antigen were injected into the cytoplasm of Xenopus oocytes, and the oocytes incubated with [32P]phosphorus‐containing medium, a 32 kDa protein was found to be preferentially phosphorylated in an NLS‐dependent manner. The incubation of fractionated cytosolic extracts prepared from mouse Ehrlich ascites tumor cells with [γ‐32P]ATP in the presence of the NLS peptides, results in the stimulation of the phosphorylation of several proteins. Similar in vitro stimulation was observed by other functional NLS peptides such as those of polyoma virus T‐antigen and nucleoplasmin. Little or no stimulation, however, was detected for peptides of mutant type and reverse type NLS of SV40 T‐antigen, and the C‐terminal portion of lamin B. Using an in vitro assay, the phosphorylation activity was fractionated chromatographically and a fraction was obtained which contained a high level of activity. The fraction was found to contain three major proteins having molecular masses of 95, 70, and 43 kDa. The in vivo and in vitro results are consistent with the existence of a protein kinase, called NLS kinase, that is specifically activated by NLS peptides.