Jun Asayama

Cardioprotective Effect of Angiotensin-Converting Enzyme Inhibition Against Hypoxia/Reoxygenation Injury in Cultured Rat Cardiac Myocytes

BACKGROUND Although ACE inhibitors can protect myocardium against ischemia/reperfusion injury, the mechanisms of this effect have not yet been characterized at the cellular level. The present study was designed to examine whether an ACE inhibitor, cilazaprilat, directly protects cardiac myocytes against hypoxia/reoxygenation (H/R) injury. METHODS AND RESULTS Neonatal rat cardiac myocytes in primary culture were exposed to hypoxia for 5.5 hours and subsequently reoxygenated for 1 hour. Myocyte injury was determined by the release of creatine kinase (CK). Both cilazaprilat and bradykinin significantly inhibited CK release after H/R in a dose-dependent fashion and preserved myocyte ATP content during H/R, whereas CV-11974, an angiotensin II receptor antagonist, and angiotensin II did not. The protective effect of cilazaprilat was significantly inhibited by Hoe 140 (a bradykinin B2 receptor antagonist), NG-monomethyl-L-arginine monoacetate (L-NMMA) (an NO synthase inhibitor), and methylene blue (a soluble guanylate cyclase inhibitor) but not by staurosporine (a protein kinase C inhibitor), aminoguanidine (an inhibitor of inducible NO synthase), or indomethacin (a cyclooxygenase inhibitor). Cilazaprilat significantly enhanced bradykinin production in the culture media of myocytes after 5.5 hours of hypoxia but not in that of nonmyocytes. In addition, cilazaprilat markedly enhanced the cGMP content in myocytes during hypoxia, and this augmentation in cGMP could be blunted by L-NMMA and methylene blue but not by aminoguanidine. CONCLUSIONS The present study demonstrates that cilazaprilat can directly protect myocytes against H/R injury, primarily as a result of an accumulation of bradykinin and the attendant production of NO induced by constitutive NO synthase in hypoxic myocytes in an autocrine/paracrine fashion. NO modulates guanylate cyclase and cGMP synthesis in myocytes, which may contribute to the preservation of energy metabolism and cardioprotection against H/R injury.

Echocardiographic measurement of right ventricular wall thickness. A new application of subxiphoid echocardiography.

The feasibility of subxiphoid echocardiography to measure the thickness of the right ventricular wall (RVWT) was investigated. In 87 (90.6%) of the 96 patients studied, adequate visualization of the echoes from the right ventricular wall was obtained using the subxiphoid technique. RVWT averaged 0.34 ± 0.08 cm (mean ± 1 SD), ranging from 0.2 to 0.5 cm in 25 normal individuals. This was not significantly different from the values in the left ventricular overload group (0.36 ± 0.10 cm). However, the RVWT was increased significantly (P < 0.001) in the combined group (0.62 ± 0.18 cm), the right ventricular (RV) pressure overload group (0.60 ± 0.13 cm) and the RV volume overload group (0.53 ± 0.11 cm). Thirty-two patients underwent diagnostic right heart catheterization which revealed a good correlation between the RVWT measured echocardiographically and the right ventricular peak systolic pressure (r = 0.84). Subxiphoid echocardiography was considered to be useful in diagnosing right ventricular hypertrophy in adults.

Energy metabolism after ischemic preconditioning in streptozotocin-induced diabetic rat hearts

OBJECTIVES The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts. BACKGROUND The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts. METHODS Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion. RESULTS In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups. CONCLUSIONS The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.

Calcium channel blockades exhibit anti-inflammatory and antioxidative effects by augmentation of endothelial nitric oxide synthase and the inhibition of angiotensin converting enzyme in the N(G)-nitro-L-arginine methyl ester-induced hypertensive rat aorta: vasoprotective effects beyond the blood pressure-lowering effects of amlodipine and manidipine.

Long-acting dihydropyridine calcium channel blockades have been shown to limit the progression of atherosclerosis and decrease the incidence of cardiovascular events in humans and animals. To investigate the vasoprotective effects beyond the blood pressure-lowering effects of these agents, amlodipine (20 mg/kg/day) and manidipine (10 mg/kg/day) were administered by gavage to NG-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats for 2 weeks. L-NAME treatment (0.7 mg/ml in drinking water) significantly decreased the gene and protein expression of endothelial nitric oxide synthase (eNOS) and increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) mRNA levels in the aorta, as determined by Western blotting and reverse transcription (RT)−polymerase chain reaction (PCR). Amlodipine and manidipine normalized the decreased expression of eNOS gene and protein, and attenuated the overexpression of NADPH oxidase, VCAM-1, and MCP-1 mRNA. Furthermore, amlodipine and manidipine prevented the L-NAME-induced increase in the angiotensin converting enzyme (ACE) mRNA content, thereby restoring control levels in the aorta. On the other hand, hydralazine treatment had no such effect in L-NAME treated rats. Furthermore, the increased expression of manganese superoxide dismutase (Mn-SOD) by L-NAME treatment was not affected by amlodipine, manidipine, or hydralazine. We concluded that the direct anti-inflammatory and antioxidative effects of calcium channel blockades in the aorta of rats with L-NAME-induced hypertension were not likely to have been mediated by the blood pressure-lowering action of these agents, but instead these beneficial effects appear to have been mediated by an augmentation of eNOS expression and by the inhibition of the expression of ACE.

Effects of ACE inhibition on myocardial apoptosis in an ischemia-reperfusion rat heart model.

Myocardial ischemia–reperfusion injury involves necrosis and apoptosis. The inhibition of angiotensin-converting enzyme (ACE) has been reported to suppress infarct size. In this study, it was investigated whether an ACE inhibitor affected myocardial apoptosis and apoptosis-related proteins in rats with experimental myocardial infarction. Anesthetized Sprague-Dawley rats were divided into four groups. Group I underwent 30 minutes of left coronary artery occlusion followed by 24 hours of reperfusion (control group); Group II underwent oral administration of the ACE inhibitor quinapril (10 mg/kg/day) before coronary occlusion (quinapril group); Group III underwent administration of the bradykinin B2-receptor antagonist Hoe 140 (250 &mgr;g/kg/day, subcutaneously) with quinapril (quinapril + Hoe 140 group); and Group IV underwent administration of Hoe 140 alone (Hoe 140 group). After reperfusion, myocardial infarct size was determined by triphenyltetrazolium chloride staining. Myocardial apoptosis was detected immunohistologically using terminal deoxynucleotidyl transferase–mediated nick end labeling staining and DNA electrophoresis. Myocardial caspase-3 activation was analyzed by Western blot and the expressions of Bcl-xL and Bax proteins were detected immunohistochemically. Quinapril significantly reduced the ratio of myocardial infarct size in the ischemic area at risk. In addition, quinapril significantly suppressed the incidence of apoptotic myocytes around the necrotic region (from 18.9 ± 0.8% to 8.6 ± 1.0%;P < 0.0001), the intensity of DNA ladder formation, and the activation of caspase-3. Hoe 140 attenuated these protective effects of quinapril. In the immunohistochemical study, Bax and Bcl-xL were expressed in myocytes, and ischemia–reperfusion abolished both proteins in the center region of ischemia. The Bax staining was equally observed among all groups. However, Bcl-xL staining remained in the ischemic area widely after quinapril treatment. In addition, Hoe 140 also depleted this effect of quinapril. These results suggest that inhibition of ACE reduces myocardial infarction and apoptosis via the bradykinin B2 receptor in part. The antiapoptotic effect of the ACE inhibitor is attributed to the changing expression of Bcl-xL.

The dual effects of nitric oxide synthase inhibitors on ischemia-reperfusion injury in rat hearts

Abstract.ObjectiveNitric oxide (NO) is known to act as a mediator of tissue injury as well as being a potent endogenous vasodilator. The functional and metabolic effects of NO on ischemia-reperfusion injury are still controversial. The aim of this study was to clarify the relationship between the degree of NO synthase (NOS) inhibition and the effects on ischemia-reperfusion injury.Methods and resultsLangendorff-perfused rat hearts were subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. The recovery of left ventricular developed pressure (LVDP), creatine kinase (CK) release, and myocardial high energy phosphates were measured in hearts perfused with or without NOS inhibitors, L-NG-monomethyl arginine (L-NMMA) or NGnitro-L-arginine methylester (L-NAME). NOS inhibitors exerted different effects on the recovery of LVDP and CK release depending on the concentration. The low dose of L-NMMA improved the recovery of LVDP, decreased the CK release during reperfusion, and preserved the myocardial adenosine triphosphate content after reperfusion. In contrast, the high dose of L-NMMA had adverse effects. L-NMMA reduced NO release in coronary effluent in a dose-dependent fashion. Both effects of L-NMMA were abolished by excessive co-administration of L-arginine and the same doses of D-NG-monomethyl arginine (D-NMMA) showed no effect on ischemia-reperfusion injury. Therefore, both effects were due to NOS inhibition. In addition, L-NMMA suppressed the myocardial malondialdehyde accumulation, an indicator of oxidative stress, which might be attributed to the beneficial effects by partial NOS inhibition. On the other hand, the high dose L-NMMA significantly decreased coronary flow during aerobic perfusion and reperfusion. Therefore, it is conceivable that the vasoactive NOS inhibition contributes to the harmful effects, which might exceed the beneficial effects due to a decrease in oxidative stress.ConclusionThe present results showed that NO inhibitors had dual effects on mechanical function and energy metabolism depending on the concentration. Non-vasoactive inhibition of NOS had beneficial effects due to the suppression of oxidative injury. However, strong vasoactive inhibition of NOS exacerbated the ischemia-reperfusion injury.

Idiopathic Enlargement of the Right Atrium

A huge saccular idiopathic dilatation of the right atrium was detected with angiography and echocardiography in a 75 year old man. Cardiac catheterization data were compared with data obtained 4 years earlier. Idiopathic right atrial enlargement is not usually accompanied by tricuspid valve disease, but in this case tricuspid insufficiency did occur over a period of 4 years, possibly as a complication in the late stage of idiopathic right atrial enlargement.Abstract Three cases of apparently massive isolated enlargement of the right atrium have been observed. A congenital malformation seems the most likely explanation in this series of cases.

Release kinetics of cardiac troponin T in coronary effluent from isolated rat hearts during hypoxia and reoxygenation

SummaryA newly developed troponin T (TnT) test for the detection of myocardial cell necrosis has been reported to be very efficient in the detection of acute myocardial infarction. The aim of the present study was to determine whether cardiac TnT in coronary effluent from isolated heart perfused with albumin-free perfusion medium could be detected using the enzyme-linked immuno-sorbent assay developed by Katus et al. Isolated rat hearts were perfused according to the method of Langendorff. Coronary flow rate was measured by timed collection of the coronary perfusate that dripped from the hearts during 5 h of hypoxia (protocol A) or 4 h of hypoxia followed by 1 h of reoxygenation (protocol B). Creatine kinase (CK) and lactate dehydrogenase (LD) levels were compared with that of TnT. Myocardial adenine nucleotides were measured by HPLC. There was a strong correlation between TnT levels in albumin-free coronary effluent and TnT levels in coronary effluent diluted 1:1 with 5% bovine serum albumin (r=0.996, N=72). The coefficients of correlation between TnT and CK or LD during hypoxia and reoxygenation were 0.891 (N=88) and 0.871 (N=88), respectively. The coefficient of correlation between CK and LD was 0.993 (N=88). There were no significant differences in either the decrease of coronary flow or the increase of TnT content between the hearts in the two protocols. There was no significant correlation between ΣTnT and energy charge of adenine nucleotides. These results indicate that cardiac TnT levels can be easily measured in albumin-free coronary effluent of isolated heart preparations. Like CK and LD, TnT is a good indicator for detecting myocardial cell damage. However, the release kinetics of TnT seem to be different than those of CK and LD. After 4 h of hypoxia, 1 h of reoxygenation has no effect on coronary flow rate or release of TnT. ΣTnT did not determine energy charge at the end of hypoxia or reoxygenation.

Release kinetics and correlation with hemodynamic dysfunction of cardiac troponin T in coronary effluent from isolated rat hearts during reperfusion.

SummaryPreviously, we reported that cardiac troponin T (TnT) can be detected and measured in coronary effluent from isolated rat hearts during hypoxia. The present study was designed to evaluate the release kinetics of TnT from post-ischemic rat hearts. Using the Langendorff technique, the hearts were reperfused for 4h after 20 min or 60 min of global ischemia. Coronary flow was measured by timing the collection of the coronary perfusate that dripped from the hearts, and left ventricular pressure (LVP) was monitored continuously during the experiments. The amount of TnT released in 1 min was compared with the release of creatine kinase (CK) and lactate dehydrogenase (LD). The release kinetics of CK and LD showed a monophasic pattern and the levels at 4 h after reperfusion returned to baseline levels. By contrast, the release kinetics of TnT showed a small peak followed by a larger and more sustained peak. There were good negative correlations between developed pressure of LVP and both Σ TnT and the amount of TnT released within 1 min at 4 h after reperfusion. These results indicate that the release kinetics of TnT is different from that of CK and LD during reperfusion, and further that cardiac TnT is a useful indicator of myocardial cell damage and can be used to evaluate the degree of myocardial cell damage in both the early and late phase of acute myocardial infarction.

Intracoronary adenosine 5′-triphosphate as an alternative to papaverine for measuring coronary how reserve

Abstract In conclusion, 50 μg of intracoronary ATP exhibited a vasodilator potency similar to that of papaverine without producing any marked changes in hemodynamics or a prolongation of the QTc. Intracoronary ATP may therefore be safer than papaverine for measuring CFR; more conclusive evidence about the safety of intracoronary ATP will have to await the conclusion of larger trials.