Tsunehiko Kuzuya

Direct association of the gap junction protein connexin-43 with ZO-1 in cardiac myocytes.

The gap junction protein connexin-43 is normally located at the intercalated discs of cardiac myocytes, and it plays a critical role in the synchronization of their contraction. The mechanism by which connexin-43 is localized within cardiac myocytes is unknown. However, localization of connexin-43 likely involves an interaction with the cytoskeleton; immunofluorescence microscopy showed that in cardiac myocytes, connexin-43 specifically colocalizes with the cytoskeletal proteins ZO-1 and α-spectrin. In transfected HEK293 cells, immunoprecipitation experiments using coexpressed epitope-tagged connexin-43 and ZO-1 indicated that ZO-1 links connexin-43 with α-spectrin. The domains responsible for the protein-protein interaction between connexin-43 and ZO-1 were identified using affinity binding assays with deleted ZO-1 and connexin-43 fusion proteins. Immunoblot analysis of associated proteins showed that the C-terminal domain of connexin-43 binds to the N-terminal domain of ZO-1. The role of this linkage in gap junction formation was examined by a dominant-negative assay using the N-terminal domain of ZO-1. Overexpression of the N-terminal domain of ZO-1 in connexin-43-expressing cells resulted in redistribution of connexin-43 from cell-cell interfaces to cytoplasmic structures; this intracellular redistribution of connexin-43 coincided with a loss of electrical coupling. We therefore conclude that the linkage between connexin-43 and α-spectrin, via ZO-1, may serve to localize connexin-43 at the intercalated discs, thereby generating functional gap junctions in cardiac myocytes.

Whole-body hyperthermia provides biphasic cardioprotection against ischemia/reperfusion injury in the rat

BACKGROUNDnHyperthermia increases cardiac tolerance to ischemia/reperfusion injury 24 hours after the heat stress. Free radicals and redox mechanisms have been implicated in such tolerance. However, the time course and its relation to the induction of antioxidative enzymes in the protection induced by whole-body hyperthermia against ischemia/reperfusion injury are unknown.nnnMETHODS AND RESULTSnHyperthermia was induced in anesthetized rats by placement in a temperature-controlled water bath. After the defined recovery interval(s) at room temperature, ischemia was induced by occlusion of the left coronary artery for 20 minutes, followed by reperfusion for 48 hours. The exposure to hyperthermia led to a recovery interval- dependent, biphasic reduction in the incidence of ventricular fibrillation during ischemia and in the size of the myocardial infarct as determined after 48 hours of reperfusion. The time course of the late-phase (24- to 96-hour recovery interval) but not the early-phase (0.5 hour) cardioprotection depended on the degree of hyperthermia. The time course of the increase in myocardial manganese superoxide dismutase (Mn-SOD) activity corresponded to that of the cardioprotective effects, although an increase in the content of Mn-SOD and of heat shock protein 72 corresponded only to the late-phase effects. Administration of an antioxidant before hyperthermia abolished the early- and late-phase cardioprotection and the increase in Mn-SOD activity.nnnCONCLUSIONSnTHe activation of Mn-SOD mediated by free radical production during hyperthermia is important in the acquisition of early-phase and late-phase cardioprotection against ischemia/reperfusion injury in rats.

Pravastatin restored the infarct size-limiting effect of ischemic preconditioning blunted by hypercholesterolemia in the rabbit model of myocardial infarction

OBJECTIVESnWe tested to find out whether pravastatin restores the infarct size (IS)-limiting effect of ischemic preconditioning (IP) and if it has any effect on the IP-induced activation of adenosine producing enzyme ecto-5-nucleotidase which plays a key role in the IP-induced cardioprotection.nnnBACKGROUNDnThe IS-limiting effect of IP is blunted by hypercholesterolemia. Recently, HMG-CoA reductase inhibitors are shown to have direct cytoprotective effects.nnnMETHODSnRabbits were fed with a normal or cholesterol (1%) added diet with or without pravastatin (5 mg/kg/day) treatment. Infarct size was measured after 30 min occlusion and 3 h reperfusion of circumflex coronary artery with or without the IP procedure (5 min occlusion and 10 min reperfusion). Additionally, ecto-5-nucleotidase activities of ischemic and nonischemic myocardium were measured immediately after IP procedure.nnnRESULTSnThis dose of pravastatin did not normalize the increased level of serum cholesterol. The IS-limiting effect of preceding IP (IS reduced from 36.7% to 9.6%, p < 0.001) was abolished by hypercholesterolemia (from 46.1% to 31.3%, p = NS) and restored by pravastatin treatment (from 35.2% to 9.4%, p < 0.001). Pravastatin treatment did not affect IS or the effect of IP under normocholesterolemia. The activation of ecto-5-nucleotidase presented as the activity ratio of ischemic to nonischemic myocardium (3.1-fold in normocholesterolemia) was blunted by hypercholesterolemia (1.8-fold, p < 0.05) and restored by pravastatin treatment (2.9-fold).nnnCONCLUSIONSnPravastatin, at the dose serum cholesterol was not normalized, restored the IS-limiting effect of IP and IP-induced ecto-5-nucleotidase activation, which were both blunted by hypercholesterolemia. The activation of ecto-5-nucleotidase may be worth further investigation as a possible mechanism for the hypercholesterolemia-induced retardation and pravastatin-mediated restoration of the cardioprotective effect of IP.

Increased expression of P-selectin on platelets is a risk factor for silent cerebral infarction in patients with atrial fibrillation : Role of nitric oxide

BACKGROUNDnPlatelet activation and decreased levels of nitrite and nitrate (NOx), stable end products of nitric oxide (NO), are reported in patients with atrial fibrillation (AF). We examined the time-course changes in plasma NOx levels and the expression of P-selectin on platelets after the onset of AF in a canine model and determined whether these parameters could be risk factors for silent cerebral infarction in patients with AF.nnnMETHODS AND RESULTSnAF was induced by rapid atrial pacing in the canine model of AF. Plasma NOx levels were significantly decreased and the levels of P-selectin on platelets and of neutrophil/platelet conjugates were significantly increased after the onset of AF in this model. The in vitro experiments demonstrated that the inhibition of NO synthesis increased the expression of P-selectin on platelets. Plasma NOx levels (19.7+/-2.4 versus 27.5+/-2.8 micromol/L) were significantly lower in 25 patients with AF compared with age- (+/-2 years) and sex-matched control subjects. Conversely, the levels of P-selectin on platelets (7.6+/-0.8% versus 4.8+/-0.7%) and of neutrophil/platelet conjugates (14.8+/-0.9% versus 8.1+/-0.6%) were significantly higher in patients with AF. Multiple regression analysis revealed that increased P-selectin on platelets and advanced age were associated with the number of foci of silent cerebral infarction.nnnCONCLUSIONSnAn irregular heart rate that is characteristic of AF appeared to blunt NO synthesis. The increased expression of P-selectin on platelets associated with the reduced NO levels was a risk factor for silent cerebral infarction in patients with AF.

Endogenous adenosine inhibits P-selectin-dependent formation of coronary thromboemboli during hypoperfusion in dogs.

The activation of platelets and the formation of neutrophil- platelet conjugates may lead to the development of thromboemboli. We studied whether blockade of adenosine receptors during coronary hypoperfusion may cause thromboemboli via P-selectin-dependent mechanisms in 30 open-chest dogs. When coronary blood flow was reduced to 20% of the control, it was stable at low levels with increases in adenosine levels. When 8-p-sulfophenyltheophylline, an adenosine receptor antagonist, was infused during coronary hypoperfusion, coronary blood flow decreased gradually and approached almost zero 20 min after its administration. Histological examination revealed thromboemboli in the small coronary vessels. During hypoperfusion in the presence of 8-p-sulfophenyltheophylline, the mAb against P-selectin attenuated both the reduction in coronary blood flow and the formation of thromboemboli, and improved contractile and metabolic dysfunction of the myocardium. Flow cytometric analysis indicated that the expression of P-selectin on platelet and neutrophil-platelet adhesion were increased during coronary hypoperfusion, and that both were further augmented by 8-p-sulfophenyltheophylline. Immunohistochemical examination showed no staining of P-selectin in the ischemic myocardium. Adenosine inhibited the thrombin-induced expression of P-selectin on platelet and neutrophil- platelet adhesion via adenosine A2 receptors. Adenosine appears to inhibit the formation of thromboemboli during coronary hypoperfusion by suppressing the expression of P-selectin on platelets and neutrophil-platelet adhesion.

A Ca channel blocker, benidipine, increases coronary blood flow and attenuates the severity of myocardial ischemia via no-dependent mechanisms in dogs

OBJECTIVESnThis study was undertaken to examine whether a dihydropyridine Ca channel blocker, benidipine, increases cardiac NO levels, and thus coronary blood flow (CBF) in ischemic hearts.nnnBACKGROUNDnBenidipine protects endothelial cells against ischemia and reperfusion injury in hearts.nnnMETHODS AND RESULTSnIn open chest dogs, coronary perfusion pressure (CPP) of the left anterior descending coronary artery was reduced so that CBF decreased to one-third of the control CBF, and thereafter CPP was maintained constant (103+/-8 to 42+/-1 mmHg). Both fractional shortening (FS: 6.1+/-1.0%) and lactate extraction ratio (LER: -41+/-4%) decreased. Ten minutes after the onset of an intracoronary infusion of benidipine (100 ng/kg/min), CBF increased from 32+/-1 to 48+/-4 ml/100g/ min during 20 min without changing CPP (42+/-2 mmHg). Both FS (10.7+/-1.2%) and LER (-16+/-4%) also increased. Benidipine increased cardiac NO levels (11+/-2 to 17+/-3 nmol/ml). The increases in CBF, FS, LER and cardiac NO levels due to benidipine were blunted by L-NAME. Benidipine increased cyclic GMP contents of the coronary artery of ischemic myocardium (139+/-13 to 208+/-15 fmol/mg protein), which was blunted by L-NAME.nnnCONCLUSIONnThus, we conclude that benidipine mediates coronary vasodilation and improves myocardial ischemia through NO-cyclic GMP-dependent mechanisms.

Amelioration by Quinapril of Myocardial Infarction Induced by Coronary Occlusion/Reperfusion in a Rabbit Model of Atherosclerosis Possible Mechanisms

BACKGROUNDnThe increased severity of the myocardial injury produced by coronary occlusion-reperfusion in models of atherosclerosis is associated with an increase in leukocyte accumulation in the ischemic myocardium. Expression of P-selectin, an adhesion molecule involved in the interaction between leukocytes and endothelium, is increased in atherosclerotic vessels. Long-term angiotensin-converting enzyme (ACE) inhibition has been shown to reduce atherosclerotic vascular change in experimental models.nnnMETHODS AND RESULTSnWe examined changes in the size of the infarct resulting from coronary occlusion/reperfusion in normally fed and cholesterol-fed rabbits that were chronically treated with quinapril. Infarct size was significantly larger in the cholesterol-fed versus normally fed rabbits. ACE activity in the ischemic and nonischemic myocardium was significantly reduced by quinapril. Chronic quinapril administration significantly ameliorated the increased myocardial injury in cholesterol-fed rabbits. Quinapril administration markedly increased the myocardial cGMP content and reduced the myeloperoxidase activity in the border region of the ischemic myocardium in cholesterol-fed rabbits. The enhanced expression of P-selectin in myocardial tissue of cholesterol-fed rabbits was also effectively reduced by quinapril treatment. The above effects of quinapril were eliminated by blockade of bradykinin B2 receptors or inhibition of nitric oxide synthesis.nnnCONCLUSIONSnChronic quinapril treatment ameliorated the severity of myocardial injury produced by coronary occlusion/reperfusion in cholesterol-fed rabbits, possibly because of reversal of the enhanced interaction between leukocytes and endothelium in the ischemic myocardium via a bradykinin-related pathway.

Inducible nitric oxide synthase augments injury elicited by oxidative stress in rat cardiac myocytes

The effects of nitric oxide (NO) produced by cardiac inducible NO synthase (iNOS) on myocardial injury after oxidative stress were examined. Interleukin-1β induced cultured rat neonatal cardiac myocytes to express iNOS. After induction of iNOS,l-arginine enhanced NO production in a concentration-dependent manner. Glutathione peroxidase (GPX) activity in myocytes was attenuated by elevated iNOS activity and by an NO donor, S-nitroso- N-acetyl-penicillamine (SNAP). Although NO production by iNOS did not induce myocardial injury, NO augmented release of lactate dehydrogenase from myocyte cultures after addition of H2O2(0.1 mM, 1 h). Inhibition of iNOS with Nω-nitro-l-arginine methyl ester ameliorated the effects of NO-enhancing treatments on myocardial injury and GPX activity. SNAP augmented the myocardial injury induced by H2O2. Inhibition of GPX activity with antisense oligodeoxyribonucleotide for GPX mRNA increased myocardial injury by H2O2. Results suggest that the induction of cardiac iNOS promotes myocardial injury due to oxidative stress via inactivation of the intrinsic antioxidant enzyme, GPX.The effects of nitric oxide (NO) produced by cardiac inducible NO synthase (iNOS) on myocardial injury after oxidative stress were examined: Interleukin-1 beta induced cultured rat neonatal cardiac myocytes to express iNOS. After induction of iNOS, L-arginine enhanced NO production in a concentration-dependent manner. Glutathione peroxidase (GPX) activity in myocytes was attenuated by elevated iNOS activity and by an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP). Although NO production by iNOS did not induce myocardial injury, NO augmented release of lactate dehydrogenase from myocyte cultures after addition of H2O2 (0.1 mM, 1 h). Inhibition of iNOS with N omega-nitro-L-arginine methyl ester ameliorated the effects of NO-enhancing treatments on myocardial injury and GPX activity. SNAP augmented the myocardial injury induced by H2O2. Inhibition of GPX activity with antisense oligodeoxyribonucleotide for GPX mRNA increased myocardial injury by H2O2. Results suggest that the induction of cardiac iNOS promotes myocardial injury due to oxidative stress via inactivation of the intrinsic antioxidant enzyme, GPX.

Reduction in infarct size by chronic amlodipine treatment in cholesterol-fed rabbits

Calcium (Ca)-dependent factors, including cholesterol-induced changes in membrane Ca permeability and Ca deposition into lesions, may contribute to plaque formation and stability during the early and late stages of atherogenesis. Amlodipine can reduce atheroma formation in cholesterol-fed rabbits and may be cardioprotective. We therefore examined the effects of chronic amlodipine treatment (5 mg/kg daily for 10 weeks, p.o.) on infarct size after 30-min coronary occlusion/48-h reperfusion in rabbits fed a diet with or without 1% cholesterol. Infarct size was significantly larger in cholesterol-fed rabbits (72.0 +/- 3.5%, n = 9, mean +/- S.E.M.) than in normal-fed rabbits (47.1 +/- 4.9%, n = 9, P < 0.05). Amlodipine treatment effectively reversed the infarct size augmentation in cholesterol-fed rabbits (46.3 +/- 6.3%, n = 9, P < 0.05), but did not affect infarct size in normal-fed rabbits (51.0 +/- 4.7%, n = 8). In both cholesterol-fed and normal-fed rabbits, Ca content and leukocyte accumulation as assessed by myeloperoxidase activity were significantly higher in the ischemic myocardium than in the nonischemic myocardium. However, Ca content and leukocyte accumulation were markedly elevated in the ischemic myocardium of cholesterol-fed rabbits compared with normal-fed rabbits. Amlodipine treatment effectively reversed this elevation. Acetylcholine showed a marked reduction in endothelium-dependent relaxation in the aorta of cholesterol-fed rabbits, which also was reversed by amlodipine treatment. These results indicate that chronic amlodipine treatment reduces infarct size only in cholesterol-fed rabbits.

Nisoldipine Selectively Induces Coronary Vasodilation and Improves Mild Myocardial Ischemia in Dogs: A Potential Role of Cellular Acidosis

We examined whether nisoldipine, a calcium (Ca) channel blocker, increases coronary blood flow (CBF) without decreasing aortic blood pressure (AoP) with ischemic and nonischemic hearts, and whether the presence of cellular acidosis in ischemic myocardium contributes to the augmentation of coronary vasodilation due to nisoldipine. In 42 dogs, coronary perfusion pressure (CPP) was reduced so that CBF decreased to 60% of the baseline, and CPP was maintained constant thereafter. First, we administered nisoldipine into a systemic vein in the ischemic and nonischemic hearts. Second, nisoldipine was administered into the canine coronary artery of the ischemic myocardium with and without administration of either sodium bicarbonate (NaHCO3), sodium hydroxide (NaOH), or amiloride. Nisoldipine (0.25–4.0 mg/kg, IV) increased CBF by 59% in the ischemic myocardium more than the nonischemic myocardium (by 34%) without reducing AoP. The infusion of nisoldipine (40 ng/kg/min, IC) increased CBF markedly by about 55% in the ischemic myocardium with increases in fractional shortening (FS; 11 ± 2% to 21 ± 2%) and lactate extraction ratio (LER; −19 ± 4% to 15 ± 2%). Increases in CBF, FS, and LER were markedly attenuated during administration of nisoldipine with concomitant administration of either NaHCO3 or NaOH. Furthermore, the extent of increases in CBF (54 ± 2 mL/100 g/min), FS (13 ± 2%), and LER (−17 ± 4%) were also markedly attenuated due to the concomitant treatment with amiloride. We conclude that myocardial cellular acidosis plays an important role in mediating coronary vasodilation affected by nisoldipine in the ischemic myocardium. H+ may modulate the property of voltage-dependent Ca channels via Na+–H+ exchange.