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The purpose of this study was to objectively assess exercise tolerance before and after cardiac valve surgery by using an objectively determined ventilatory anaerobic threshold (AT). Nine patients (mean age: 38.2 +/- 8.1 years) with predominantly mitral regurgitant lesions were studied by a symptomatic maximal treadmill exercise test which included a determination of AT. The mean lengths of time from preoperative exercise testing to cardiac surgery, and from surgery to postoperative exercise testing were 5.9 +/- 4.0 and 12.1 +/- 8.3 months, respectively. The determination of AT on data plots was performed after blinding to patient identification and pre- vs postoperative status. After surgery, the clinical symptoms and NYHA class improved significantly with a decrease in the cardio-thoracic ratio and echocardiographic diastolic dimensions. The mean peak VO2 (ml/kg/min) increased significantly from 20.2 +/- 7.1 to 29.7 +/- 7.9 (p < 0.01). Together with these changes, AT (ml/kg/min in VO2) increased from a mean of 14.8 +/- 4.8 to 22.8 +/- 5.5 (p < 0.01). In conclusion, symptomatic improvement and an increase in peak oxygen uptake after cardiac valve surgery were accompanied by a significant increase in the objectively determined AT. AT determined in a blind manner provides an objective means of evaluating exercise tolerance when a double-blind intervention cannot be performed.

BACKGROUND There have been no reports on the presence of the tissue renin-angiotensin system in the human heart, although the presence of angiotensinogen has been described in the animal heart. METHODS AND RESULTS To determine whether angiotensinogen is synthesized in the human heart, we examined angiotensinogen messenger RNA (mRNA) synthesis in autopsy hearts by using ribonuclease protection assay. As a result, angiotensinogen mRNA was detected in the atrial muscle, muscles of the conduction system, and the left ventricular wall. In the left ventricular wall, mRNA expression was more prominent in the subendocardial muscles than in the midcardial or epicardial muscles. Using a monoclonal antibody to human angiotensinogen in immunoblotting experiments, we detected two closely spaced bands at approximately 70 kd in the heart, which was quite consistent with the human angiotensinogen molecule. Immunohistochemical studies with this monoclonal antibody demonstrated intense immunoreactivity in the atrial muscles, the muscles of the conduction system, and those of the subendocardial layers. CONCLUSIONS We conclude that angiotensinogen was synthesized in the human heart. It was evident that the localization of angiotensinogen was not ubiquitous in the cardiac muscles, showing its predilection for the atrial muscles, muscles of the conduction system, and subendocardial layer of the left ventricle.

The incidence of cardiac events in Japanese men (mean age 54 +/- 10 years) with atypical or nonanginal chest pain was assessed prospectively. Three groups of patients, those with typical angina (TA; n = 134), those with atypical angina (AA; n = 192), and those with nonanginal chest pain (NA; n = 311) were studied with regard to cardiac events (cardiac death or nonfatal myocardial infarction), risk factors, and results of exercise testing. The total cholesterol and high-density lipoprotein-cholesterol levels were significantly different among the three groups, but no differences were observed for other risk factors. The incidences of cardiac events were 8.5%, 2.2%, and 1.3% in the TA, AA, and NA groups, respectively (p less than 0.05) during 3.2 +/- 1.5 years. Significant ST segment changes were observed in 70.1%, 25.5%, and 18.0% of the patients in the TA, AA, and NA groups, respectively, at the time of enrollment. The risk ratio for cardiac events in a positive exercise test was 1.7 (difference not significant) for the TA group and 6.1 (p less than 0.05) for the others combined (AA and NA groups). The risk ratio adjusted for risk factors (Cox model) was 2.2 (difference not significant) for the TA group and 4.9 (p less than 0.05) for the AA and NA groups combined. In conclusion, the incidence of cardiac events in those with AA or NA chest pain was relatively low in Japan, but the independent prognostic significance of positive exercise testing was demonstrated in this group of patients.

Continuous production of endothelium-derived nitric oxide (NO) in peripheral vessels has been shown to modulate vascular resistance and blood pressure. NO is also formed in the brain upon activation of glutamate receptors, which are thought to mediate central autonomic reflexes. In the present study we assessed whether NO plays a role in central autonomic regulation. For this, we have investigated the effects of NG-methyl-L-arginine (NMA), a selective inhibitor of NO synthesis from L-arginine, on sympathetic renal nerve activity (RNA), blood pressure, and heart rate in the anesthetized rat. NMA elicited a dose-dependent sustained increase in blood pressure (approximately 20 and 30 mm Hg, 5 minutes after 10 and 50 mumol/kg i.v., respectively). Heart rate and RNA decreased transiently (15 beats per minute and 40%, respectively); RNA subsequently increased (100%) while blood pressure remained elevated. Baroreceptor deafferentation markedly altered these responses to NMA; the transient decreases in heart rate and RNA were abolished, whereas the increases in RNA and blood pressure were significantly potentiated. After spinal C-1-C-2 transection, there was no increase in RNA, and blood pressure increased to a smaller extent. L-Arginine blocked the NMA-induced increases in blood pressure and RNA. Thus, in addition to modulating vascular resistance by a peripheral action, NO may also play a role in the central regulation of sympathetic tone.

We assessed the effect of stopping smoking on future cardiac events (cardiac death and non-fatal myocardial infarction (MI)) in 90 male smokers with previous MI by a prospective follow-up study. At the time of enrollment, the patients were divided into 2 groups according to their current smoking status (Group A: those who had stopped smoking (n = 60), and Group B: those who were still smoking (n = 30)). Follow-up was performed prospectively by annual postal questionnaires. During the mean follow-up period of 3.1 +/- 1.4 years, 13 cardiac events (11 cardiac deaths and 2 cases of non-fatal MI) occurred in the 87 evaluable patients. The cardiac event rates in Groups A and B were 8.5% and 28.6%, respectively, and the relative risk was 3.4 (Group A vs B, p less than 0.05) by univariate analysis. The relative risk using multivariate analysis (Cox) was also statistically significant (3.1, p less than 0.05). HDL-cholesterol was significantly lower in Group B than in Group A, but other coronary risk factors apart from smoking were not significantly different between the two groups. At the end of the follow-up period, 89.8% of the patients in Group A remained nonsmokers and 21% of the patients in Group B had stopped smoking.Stopping smoking significantly reduced the risk of future cardiac events in male smokers with previous MI. This prospective follow-up study demonstrated that a significant reduction in the risk of future cardiac events could be achieved by stopping smoking in this group of patients.

The purpose of this study was to construct a simple ergometer for the 31P NMR spectroscopic study of dynamic forearm exercise in a whole body magnetic resonance imaging system and to evaluate the total system and the physiological response to this type of exercise using a multistage protocol.The system consisted of a completely nonmagnetic assembly including a rope, pulley and weights. The work of lifting weights was quanti tated. The exercise protocol of 1-min increments in work load enabled subjects to reach maximal effort. Phosphocreatine decreased linearly with an increase in work load and was accompanied by a fall in pH and an increase in lactate level in the antecubital vein of the exercising fore arm; concomitantly, there was a slight increase in whole body oxygen uptake and heart rate. Spectroscopy gave reproducible results using this exercise protocol.These results demonstrate that this system provides a reliable means for performing 31P magnetic resonance spectroscopy studies during fore arm exercise.

The aim of this study was to investigate the correlation between hypoxic myocardial cell injury and intracellular protease activity. Cardiac myocytes were isolated from neonatal rat hearts and cultured in Eagle's modified minimum essential medium. Myocytes were incubated in hypoxic conditions for 6 hours. The cell death rate during hypoxia rose to 80% after 6 hours. Extracellular protease activity was elevated to 4 units during hypoxia, much higher than the 0.7 units in aerobic states at 6 hours. This extracellular protease activity in hypoxic conditions was markedly inhibited by leupeptin and EDTA, and weakly inhibited by the cysteine protease inhibitor, NCO-700, but phenylmethyl sulfonyl fluoride did not inhibit the protease activity. To identify the protease activated during hypoxia, calpain-specific inhibitors were added to the incubation mixture. Calpain inhibitor 1 and calpastatin, an endogenous selective calpain inhibitor, markedly inhibited extracellular protease activity during hypoxia. NCO-700 also inhibited intracellular protease activity. NCO-700 reduced hypoxic cell death to 30% after 6 hours of hypoxy-genation. These observations indicate that calpain is activated during hypoxia and leads to irreversible cell membrane degradation after 6 hours of hypoxygenation.

Cell death during hypoxia rose to 80% after 6 h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6 h. This activity was markedly inhibited by calpain-specific inhibitor I ( n -acetyl-leucyle-leucyle-norleucinal). β-Adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. On the other hand, α-adrenergic blocking agents did not affect calpain activity and cell death under hypoxic conditions. These results prove that β-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.

We observed that in hypoxic myocardial cells prostacyclin and arachidonic acid release increased and that during hypoxia phospholipid degradation also occurred. In order to clarify the mechanism of phospholipid degradation, we determined the activity of phospholipases A 2 and C. We found that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were markedly decreased and that lysophosphatidylcholine and lysophosphatidylethanolamine were increased. In contrast, there was only slight phosphatidylinositol degradation and no lysophosphatidylinositol elevation was observed. These results show that phospholipase A 2 was activated in hypoxic myocytes and had substrate specificity towards PC and PE. To study phospholipase C activity, membrane phospholipids were labeled with [ 3 H]choline, [ 3 H]inositol or [ 3 H]ethanolamine. The release of inositol was observed, but neither choline nor ethanolamine was released. In hypoxia, myocardial-cell phospholipase C has high substrate specificity towards phosphatidylinositol. The activation of phospholipases is closely related to the intracellular Ca 2+ concentration; it is thought that inositol polyphosphatides may regulate intracellular Ca 2+ . We determined how Ca 2+ influx occurs in hypoxia. β-Adrenergic blockade and Ca 2+ antagonists markedly suppressed Ca 2+ influx, phospholipase A 2 activity, phospholipase C activity and cell death. However, the α 1 -adrenergic blockade was less effective in suppressing these phenomena. These results suggest that in hypoxic myocardial cells Ca 2+ influx mediated by β-adrenergic stimulation activates phospholipases A 2 and C, and that phospholipid degradation and prostacyclin release then occur.

The effect of beta-blockade in dilated cardiomyopathy was studied by a questionnaire survey. Thirty-three cases were monitored in whom metoprolol (22 patients, 35.9 +/- 20.4 mg, mean +/- SD), propranolol (four patients, 26.3 +/- 7.5 mg), or other beta-blockers (seven patients) were administered. Four patients died, but no direct relationship was found between administration of beta-blocker and death. The NYHA functional class improved significantly. The mean heart rate decreased from 96/min to 77/min (P less than 0.01). The mean cardiothoracic ratio decreased from 55.6% to 52.1% (P less than 0.01). The mean ejection fraction of the left ventricle measured by echocardiogram increased from 30.4% to 36.9% (P less than 0.01). Exercise tolerance in the treadmill test improved significantly. There was no change in blood pressure, nor were there arrhythmias seen on Holter electrocardiograms. In two patients, congestive heart failure deteriorated after administration of beta-blockers. It is concluded that beta-adrenergic blockade has a beneficial effect in most of the patients with dilated cardiomyopathy.

We studied the effects of elastase on [3H]thymidine incorporation into aortic smooth muscle cells. When elastase was added to cultured aortic smooth muscle cells, [3H]thymidine incorporation was inhibited in a dose-dependent manner in the presence of fetal bovine serum. Elastase also inhibited this incorporation in cells treated with epidermal growth factor. Epidermal growth factor (50 ng/ml) stimulated thymidine incorporation, without elastase, but with the addition of 20 units/ml of elastase the incorporation was inhibited 70%. The incorporation of thymidine into cells treated with 50 ng/ml epidermal growth factor was also inhibited 50% by a low concentration of elastase (5 units/ml). These inhibitory effects on thymidine incorporation were also observed in cells stimulated with platelet-derived growth factor. Platelet-derived growth factor (20 units/ml) markedly stimulated thymidine incorporation into cells, and elastase inhibited its activity in a dose-dependent manner. These results suggest that elastase has the potential to prevent the development of atherosclerosis by inhibiting smooth muscle proliferation.

There were remarkable differences in the serum total and unbound concentrations of disopyramide (DP) and mono-N-dealkyldisopyramide (MND), the major metabolite of DP, among patients with arrhythmias. Serum levels of alpha 1-acid glycoprotein (AAG) also varied among the same patients. To predict the unbound DP concentrations, we have obtained the serum concentration-time curves of unbound DP in these patients by means of total DP concentration, DP pharmacokinetic parameters, AAG levels and dissociation constants of DP and MND at specific AAG binding sites. In patients with normal AAG levels, the theoretical values of unbound DP were in good agreement with the measured concentrations. On the other hand, in patients with high AAG levels, the theoretical values could be obtained by correcting the calculated values using both AAG levels of each patient and the equation correlating the unbound DP fraction to AAG concentrations. These findings indicate that patient AAG levels can provide valuable information permitting the rapid estimation of unbound DP concentrations, the pharmacologically active fraction, and the development of effective DP dosage regimens.

Cardiac membranes from 26-, 52- and 104-week-old Wistar rats were used to investigate the age-related alterations in the beta-adrenergic receptor-adenylyl cyclase system. The densities and affinities of beta-adrenoceptors did not change with aging. There were no significant changes in the total amount of stimulatory G-protein (Gs), and in Gs activity measured in a reconstitution assay using human platelet membranes. The major isoform of Gs alpha, however, changed from a 45,000 to 52,000 dalton peptide with aging. The total amount of pertussis toxin substrates (Gi2 and Go) decreased significantly with aging. This finding was supported by the fact that pertussis toxin-induced potentiation of adenylyl cyclase activity was markedly reduced in the aged group. The activity of catalytic protein assessed by forskolin-stimulated adenylyl cyclase activity was decreased at 104 weeks. On the other hand, GTP analogue-stimulated adenylyl cyclase activity was significantly potentiated in the same group. These results suggest that the decreased sensitivity to catecholamines observed in aged hearts is mainly due to a dysfunction of catalytic protein, and that decreased Gi activity partially compensates for this catalytic dysfunction.

This study was undertaken to investigate the correlation between hypoxic cell injury and protease activity, as well as the effects of alpha 1-, and beta-adrenergic blocking agents and calcium antagonists during hypoxia. Cell death during hypoxia rose to 80% after 6h. Calpain activity increased to 4 units during hypoxia, much higher than the 0.7 units seen in aerobic condition at 6h. This activity was markedly inhibited by calpain-specific inhibitor I (n-acetyl-leucyle-leucyle-norleucinal). alpha 1-adrenergic blocking agents did not affect calpain activity and cell death under hypoxia. On the other hand, beta-adrenergic blocking agents and calcium antagonists suppressed the calpain activity and decreased cell death during hypoxia. These beta-adrenergic blocking agents and calcium antagonists also inhibited intracellular calcium-influx during hypoxia. These results suggest the beta-adrenergic receptor stimulation activates adenylate cyclase activity and enhances calcium-influx during hypoxia. The elevated intracellular calcium concentration then stimulates calpain activity under hypoxia. These results prove that beta-adrenergic blocking agents and calcium antagonists prevent protein degradation during hypoxic cell injury.

We studied the effects of atrial natriuretic factor (ANF) on the conversion of angiotensin I to angiotensin II. Pulmonary artery endothelial cells converted 1.22 nmol/min per dish [125I]angiotensin I to angiotensin II, but ANF suppressed the conversion by 0.475 nmol/min per dish. The maximum rate of inhibition of angiotensin converting enzyme (ACE) activity by ANF was 60% at 10(-6) mol/l ANF compared with control conditions. The amino acid fragments of ANF were studied to determine whether its specific structure was necessary for inhibiting ACE in endothelial cells. We found that atriopeptin II (fragment 5-27) inhibited ACE activity as much as ANF. Fragment 7-25 had the same rate of inhibition, but fragment 13-27 had no effect on the conversion of angiotensin I to angiotensin II. These results suggest that the amino acid sequence of Cys (7)-Cys (23) in ANF is important for the inhibition of ACE in pulmonary endothelial cells.

In the present experiment, we demonstrated that IP3 kinase activity was increased in SHRSP heart compared to WKY heart. IP3 kinase activity in the heart was highest in the cytosolic fraction in both SHRSP and WKY. Its activity progressively increased with age in 5- to 20-week SHRSP. The activity reached about three times the level of 5-week-old SHRSP in 40-week-old SHRSP. On the other hand, in WKY it was 1.3-fold at 40 weeks compared with that at 5 weeks. We determined the effect of divalent cations on IP3 kinase activity. Ca2+ stimulated its activity in a dose-dependent manner at 10(-9) to 10(-6) M. In SHRSP it was enhanced about 2.1-fold at 10(-6) M of Ca2+, but in WKY it was 1.5-fold at 1.0(-6) M of Ca2+. Mn2+ also stimulated IP3 kinase activity in both groups of animals, while, Fe2+, Zn2+, and Cu2+ inhibited IP3 kinase activity. In our experiment IP3 kinase activity was increased in SHRSP and its activity was markedly affected by divalent cations. These data suggest that the accumulations of IP3 and IP4 after hormonal stimulation play a physiologic role, possibly by alteration of Ca2+ levels in cardiac tissue.

We studied the effects of platelet activating factor (PAF) on angiotensin-converting enzyme (ACE). PAF (1 · 10 −10 to 1 · 10 −6 M) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 1 nmol/dish of 125 I-angiotensin I to angiotensin II in the absence of PAF. ACE activity was increased to 2.5 nmol/dish by the addition of 1 · 10 −6 M of PAF. To clarify the mechanism of this stimulatory effect of PAF on ACE, Ca 2+ influx and inositol 1,4,5-trisphosphate (IP 3 ) release in pulmonary artery endothelial cells were determined. PAF stimulated Ca 2+ influx in a dose-dependent manner. PAF also stimulated phospholipase C (PLC) activity and released IP 3 . To study the relationship between PLC activity and ACE activity, neomycin was added. The Ca 2+ influx and IP 3 release stimulated by 10 −6 M of PAF were suppressed by about 60–70%. ACE activity was also inhibited up to 70% in the presence of PAF (10 −10 − 10 −6 M) by 50 M of neomycin. These results suggest that ACE was stimulated by PAF, and that its activityl in endothelial cells may be mediated by the PI-turnover pathway via changes in PLC activity and IP 3 -mediated Ca 2+ release from intracellular stores.

We have reported that low doses of endothelin-3 (ET-3) elicited continuous vasodilation of rat mesenteric arteries, which is possibly related to endothelium-derived relaxing factor (EDRF). In order to clarify whether or not the vasodilating effects of ET-3 are associated with EDRF, we examined the effects of L-NG-monomethyl arginine (L-NMMA), an analog of L-arginine, on low-dose ET-3 induced vasodilation of rat mesente-Hc arteries. Infusion of 50 microM L-NMMA inhibited the vasodilation induced by 10(-13) M ET-3 and rather elicited an increase in perfusion pressure, which itself was decreased by infusion of 150 microM L-arginine. In the presence of 50 microM L-NMMA, 10(-13) M ET-3 did not elicit any vasodilation of the mesenteric arteries preconstricted with NE, in which 150 microM L-arginine, but not D-arginine, caused considerable vasodilation. These data suggest that the vasodilating effects of low doses of ET-3 are associated with EDRF as an endothelium-derived nitric oxide.

In the cooperative, multi-center trial, 491 medically treated patients with coronary artery disease were thoroughly assessed and followed for an average of 49 months. The rate of cardiac death and/or non-fatal myocardial infarction was 9.8%; 64% of cardiac events were fatal. Three factors, such as number of diseased coronary arteries, other diseased organs and the cardiothoracic ratio, contributed significantly to the evolution of cardiac events. The outcome of cardiac events in Japan in the period from 1973 to 1985 in patients with coronary artery disease was more favorable than those reported recently in the United States. The results of this study will serve as a control data base for further studies in Japan.

Varying values for the acetylcholine (ACh) concentration in the rat heart have been reported. The possibility that the method of sampling may influence prompted a comparison of heart levels of ACh obtained by two different procedures for sacrificing animals. One method was by microwave irradiation in vivo and the others being in vitro on the irradiated heart removed after decapitation. There were significant differences found in cardiac ACh concentration between the in vivo irradiated group and the decapitation groups. In decapitated animals, the cardiac ACh concentration became increasingly lower on standing. We also measured the ACh concentration of right atrium, left atrium, right ventricle and left ventricle. They were 4.62 +/- 1.57 nmol/g (mean +/- SD), 2.58 +/- 1.01, 2.76 +/- 1.00 and 2.12 +/- 0.70, respectively. We conclude the microwave irradiation in vivo is a more appropriate method for determining the cardiac ACh concentration.

Electromechanical responsiveness to angiotensin II (Ang II) receptor stimulation in ventricular myocardium and myocytes of hypertrophic cardiomyopathic Syrian hamsters (BIO 14.6) was examined and compared with that in preparations of normal hamsters (F1B) using conventional microelectrode and patch clamp techniques. Action potential duration (APD) and developed tension (DT) corrected for the cross-sectional area of the papillary muscles of 14-20 week-old BIO 14.6 hamsters were significantly smaller than those in preparations of age-matched normal hamsters. An Ang II (1 microM)-induced increase in DT in BIO 14.6 papillary muscles (24.7 +/- 11.0%) was significantly smaller than that in F1B papillary muscles (53.8 +/- 8.5%), which was associated with a smaller increase in APD in BIO 14.6 papillary muscles. In ventricular myocytes of both BIO 14.6 and F1B hamsters. Ang II increased the calcium current (ICa) following a transient decrease in ICa. However, the magnitude of the Ang II-induced increase in ICa in BIO 14.6 myocytes (35.5 +/- 7.5%) was significantly smaller than that in F1B myocytes (86.0 +/- 19.7%), suggesting a causal relationship between ICa and mechanical response to Ang II in these hamsters. The depressed responsiveness to Ang II receptor stimulation in hypertrophic cardiomyopathic hamster is in a marked contrast with the enhanced responsiveness to alpha 1-adrenergic stimulation, which was demonstrated by previous studies, and may be one of adaptational changes to the activated renin-angiotensin system in the cardiomyopathy.

To identify genetic factors in the immune system which control the susceptibility to dilated cardiomyopathy (DCM), HLA class II DNA typing was performed in 61 Japanese patients, using PCR/SSO probe analyses. The frequencies of HLA-DQB1*0503 (15% vs 5%; RR = 3.06, chi 2 = 7.19) and DQB1*0604 (21% vs 10%; RR = 2.41, chi 2 = 6.20) were significantly increased and that of HLA-DQB1*0502 (RR = 1.74) was slightly increased in the DCM patients. The frequency of DQB1*0303 (16% vs 31%; RR = 0.44, chi 2 = 5.16) was significantly decreased in the patients. The increased HLA-DQB1 alleles have a histidine residue in common at the 30th codon for the HLA-DQ beta chain. Among the genetic markers studied by Southern blot analyses, IGLV (immunoglobulin lambda light chain, pV3.3) showed a strong association with DCM, i.e. A2/A2 genotype was found in 37.7% of patients whereas it was observed in only 18.9% of the control subjects (RR = 2.6, chi 2 = 7.77). The frequency of this genotype was higher in patients under age 45 years at the time of diagnosis (45.5%, RR = 3.6, chi 2 = 10.02). These results suggest that HLA and immunoglobulin genes are closely linked to susceptibility to DCM.

We demonstrated that phosphatidylinositide-specific phospholipase C (PLC) activity was greater in cardiomyopathic hamster hearts (BIO 14.6 and BIO 53.58) then in hamster controls (F1b). Inositol trisphosphate (IP3) production was markedly greater in both of the cardiomyopathic hamsters, BIO 14.6 and BIO 53.58. We have also determined the sarcoplasmic reticulum (SR) function of heart. Calcium uptake into SR markedly increased in BIO 14.6. On the other hand, it significantly decreased in BIO 53.58 compared with F1b. It is well known that IP3 stimulates calcium release from SR. In BIO 14.6, calcium relrease from SR stimulated by IP3 increased, but its effect decreased in BIO 53.58 compared with F1b. These results suggest that PI response may produce high intracellular calcium levels in both BIO 14.6 and BIO 53.58 myocytes. In addition, in the BIO 53.58 hamster the sarcoplasmic reticulum deteriorate in function. It was concluded from these results that a prolonged high intracellular calcium level may lead to the death of BIO 53.58 myocytes. The expression of angiotensinogen mRNA was observed in the hamster heart. There was no differences in its expression level between F1b, BIO 14.6 and BIO 53.58. There was no effect of ages on its expression in these hamster hearts. We have also determined the distribution of angiotensinogen in these hamsters. At 4 weeks of age, the immunohistochemical study revealed that angiotensinogen was widely distributed in subendcardium in these hamsters. There was no difference in its distribution between F1b, BIO 14.6 and BIO 53.58. But at 20 weeks old of age its immunoreactivity decreased in BIO 53.58. There was no effect of age on its reactivity in F1b and BIO 14.6. We have detected angiotensinogen in heart, but its role is still not clear. A further study should be done to clarify its role in cardiac hypertrophy and cell damage.

It is well known that failing hearts show diminished responsiveness (desensitization) to catecholamines. In this study, 2 different animal models were used to investigate the alterations in the hormone sensitive cardiac adenylyl cyclase system in a congestive heart failure. In the first model, cardiomyopathic Syrian hamsters (BIO53.58), we found reduced activity of the catalytic protein of adenylyl cyclase; this reduction was more prominent in an older animals (28-week-old vs 16-week-old). At both ages, the amount of inhibitory GTP-binding protein (Gi) was markedly increased in BIO53.58 compared to control healthy hamsters. Moreover the increased Gi was shown to be fully functional in the inhibitory pathway of the adenylyl cyclase system. In the second model, chronically norepinephrine-infused rats, we found a decrease in beta-adrenergic receptor density at an early stage of injection (3 days), while the activity of catalytic protein decreased beyond 14 days of injection, and the amount of Gi increased after 7 days of injection. These results suggest that increased plasma catecholamine concentrations in the setting of congestive heart failure might be a major trigger for qualitative and quantitative alterations observed in various components of cardiac adenylyl cyclase system, and that GTP-binding proteins and catalytic protein of adenylyl cyclase are involved in the mechanism of desensitization especially after chronic in vivo stimulation of adrenergic receptors.

The accumulation of both Inositol-(1,4,5)-trisphosphate (IP3) and Inositol-(1,3,4,5)-tetrakisphosphate (IP4) after hormonal stimulation has a physiological role, possibly in altering Ca2+ levels in cardiac tissue. However, the accumulation of inositol polyphosphate under pathophysiological conditions has not been studied. In our experiments the metabolism of phatidylinositol and IP3 in cardiac myocytes as investigated. It was shown that basal levels of cytosolic phosphatidylinositol specific phospholipase C (PI-PLC), phosphatidylinositol-(4,5)-bisphosphate specific phospholipase C (PIP2-PLC) activities markedly increased in stroke-prone spontaneously hypertensive rats (SHRSP) with age compared with age matched Wistar Kyoto rats (WKY). IP3 kinase and IP3 phosphatase activities also increased in SHRSP hearts with age. Their activities increased in WKY, but to a lesser extent than in SHRSPs. These data suggest that a PI turnover pathway such as the phosphatidylinositol 4,5-bisphosphate-IP3-Ca2+ pathway or the diacylglyceride-protein kinase C pathway may have an important role in the development of hypertrophy in SHRSP heart.

The renin-angiotensin system acts to constrict vessels and enhance renal retention of sodium and water to raise the blood pressure. Angiotensinogen (ang-n) is synthesized in the liver and released into the blood. It is cut by renin, which is produced by the kidneys, and becomes angiotensin I. Angiotensin I is cleaved by angiotensin converting enzyme (ACE) into angiotensin II, which is an active pressor substance. Recently, the presence of cardiac renin-angiotensin system has been reported in some animals [1,2,3] but it has not been investigated fully in the human heart. Angiotensin II appears to be a stimulus for cardiac hypertrophy [4]. In clinical studies, ACE inhibitor has been reported to cause regression of cardiac hypertrophy [5]. Bearing these findings in mind, we studied the presence of ang-n, renin mRNAs and ang-n protein in human autopsy hearts and discussed its relation to cardiac hypertrophy.

Various diseases and clinical settings can induce congestive heart failure (CHF). As such, it is unlikely that a single mechanism can explain the cardiac dysfunction observed in CHF. However, CHF patients (or animals) share several similar features: reduced cardiac contractility, increased sympathetic nerve activity, and activation of several hormonal systems (e.g., renin-angiotensin system, vasopressin, and atrial natriuretic factor) to compensate for the impaired cardiac function. In this setting, cardiac receptor, GTP-binding proteins, and their effector molecules (adenylyl cyclase, phospholipase C, several ion channels) are inevitably exposed to chronically elevated neurohormonal stimulation. A widely recognized concept is that a chronic increase in such stimulation can desensitize target cell receptors and post-receptor signal transducing pathways (1). It is therefore likely that in CHF one or more types of desensitization contributes to a reduction in signal transducing efficiency in order to maintain intracellular homeostasis. To date, several laboratories have reported a decrease in β-adrenergic receptor (βAR) density in CHF patients and model animals (2–4, 5 for review). Recently, reports of several studies have indicated that the inhibitory GTP-binding protein (Gi) can be increased in CHF patients (6,7) and animal models (8). Although direct evidence for a change in the catalytic protein of adenylyl cyclase has not been available, limited information have suggested a reduced catalytic activity in terminally failing hearts (9). In this study, we have suggested changes of βAR, GTP-binding protein and catalytic protein using two different animal models with an emphasis on defining the kinetics for the desensitization process of adenylyl cyclase system.

In cardiac hypertrophy,collagen accumulation in the myocardial interstitium is associated with hypertrophy of cardiomyocytes [1]. Collagen remodeling causes impaired ventricular compliance,abnormal electrical conduction and impaired oxygenation of the cardio-myocytes [2].At present,factors that regulate cardiac collagen metabolism are not elucidated.Eghbali et al [3] found that cardiac fibroblasts are mainly responsible for the synthesis of fibrillar type I and III co11agen.Recent studies have demonstrated that angiotensin II (A II),in addition to its inotropic and chronotropic actions,may exert effects by acting as a growth factor in the car¬diovascular system and that there may be local autocrine or paracrine renin-angiotensin system in several tissues [4].There are no data concerning the effects of A II on cardiac nonmyocytes.In this study we examined the effects of A II on collagen synthesis by cardiac fibroblasts in normotensive and hypertensive rats.

Plasma membrane inositol phospholipids were breakdown by hormones and neurotransmitters through the mediation of the specific receptors (1–4). This phosphatidylinositol (PI) turnover pathway generates two second messengers, inositol-(l,4,5)-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG) (5,6). DAG stimulates membrane-bound phospholipid-dependent, Ca2+- dependent protein kinase C (7), while IP3 releases Ca2+ from stores in the sarcoplasmic reticulum (8,9). The physiological significance of this Pi–turnover pathway is not still clear in any mammalian cell system. It is controversial whether IP3 stimulates Ca2+ release from SR in cardiac myocytes.

Hypertrophic cardiomyopathy (HCM) is a clinically and genetically heterogeneous disease. Nearly half of HCM shows familial occurrence wit hautosomal dominant inheritance. To investigate the causative genes of familial HCM, we performed linkage analyses by using RFLP (Restriction Fragment Length Polymorphism) method. In addition, we used SSCP(Single-strand Conformation Polymorphism) method to detect missense mutations within the beta cardiac myosin heavy chain (βMHC)which is known to be one of the causative genes for familial HCM.

It has been demonstrated that components of the renin-angiotensin system such as renin, angiotensinogen (ATN), angiotensin-converting enzyme (ACE) and angiotensin II receptor exist within the heart and function independently from circulating renin-angiotensin system [1–4]. The potent myotropic actions of angiotensin II, as well as its effects on positive inotropism and chronotropism, make it likely that the heart is the site of a locally active renin-angiotensin system. It is suggested that angiotensin II is one of the contributing factors to regulation of cell growth, moreover, ACE inhibitors regress left ventricular hypertrophy. The purpose of this study is to determine whether intrinsic renin-angiotensin system play a role for the development and the regression of left ventricular hypertrophy in spontaneously hypertensive rats (SHR).

To assess exercise energy metabolism of forearm flexor muscles in rowers, six male student rowers and six control subjects matched for age and sex were studied using phosphorus-31 magnetic resonance spectroscopy (31P-MRS). Firstly, to adjust for the effect of differences in cross-sectional muscle area, the maximal cross-sectional area (CSAmax) of the forearm flexor muscles was estimated in each individual using magnetic resonance imaging. Multistage exercise was then carried out with an initial energy production of 1 J · cm−2 CSAmax for 1 min and an increment of 1 J · cm−2 CSAmax every minute to the point of muscle exhaustion. A series of measurements of31P-MRS were performed every minute. The CSAmax was significantly greater in the student rowers than in the control subjects [19.8 (SD 2.2) vs 17.1 (SD 1.2) cm2,P

We demonstrated that the activities of phosphatidylinositide-specific phospholipase C, inositol 1,4,5-trisphosphate (IP3) kinase, and IP3 phosphatase were enhanced in cardiomyopathic hamster hearts (BIO 14.6 and BIO 53.58) in comparison to control hamsters (F1b). Release of both arachidonic acid and prostacyclin was markedly enhanced by norepinephrine in the cardiomyopathic hamsters. Phospholipase C in heart has high substrate specificity to phosphatidylinositol. IP3 production was markedly enhanced in the cardiomyopathic hamsters. We also determined the intracellular calcium concentration, which was higher in BIO 53.58 hamsters than in BIO 14.6 hamsters at 5-20 weeks of age. There was no significant difference in the intracellular calcium level between F1b and BIO 14.6 hamsters at 5 weeks of age. These results suggest that phosphatidylinositol turnover stimulated by norepinephrine may produce high intracellular calcium levels in both BIO 14.6 and BIO 53.58 myocytes. In addition, in BIO 53.58 hamsters, some mechanism such as the sarcoplasmic reticulum, which controls the intracellular calcium level, may deteriorate in function. We concluded from these results that a prolonged high intracellular calcium level may lead to the death of BIO 53.58 myocytes.

Cultured vascular endothelial cells were exposed to fluid shear stress by means of a rotary-disc shear-loading device, and the physiological effects of the conditioned medium (CM) and the homogenate (HM) of the cells on migration, adhesion and growth of endothelial cells (EC) or smooth muscle cells (SMC) were studied. Effects of shear stress on the production and secretion of collagen, one of the extracellular matrices of EC, were also studied. CM stimulated the adhesion and growth of SMC, but not of EC themselves. The ability to stimulate SMC adhesion and growth was similar in CM obtained from the static and shear-loaded cells. HM of the shear-loaded EC stimulated SMC migration. Further, HM of the shear-loaded EC contained increased amounts of collagen compared with the static EC. These results suggest that: 1) EC produce and secrete accelerators for the adhesion and growth of SMC, 2) EC react to the physical stimulus of fluid shear stress to produce stimulators of SMC migration, and 3) EC produce collagen, the production of which is enhanced by fluid shear stress.

The effects of increased free fatty acid (FFA) levels on ventricular arrhythmias remain controversial. Using ventricular fibrillation threshold (VFT), we examined the relationship between FFA levels and ventricular arrhythmias. Isolated rat hearts were perfused with palmitate bound to either albumin or fatty acid binding protein (FABP) by Langendorf's method. The VFT was determined by electrical stimulation. Perfusion with 0.12 mM albumin alone, 0.12 mM palmitate bound to 0.12 mM albumin, and 0.36 mM palmitate bound to 0.12 mM albumin did not lower the VFT significantly. However, 0.60 mM palmitate bound to 0.12 mM albumin lowered VFT from 2.19 +/- 0.20 mA to 1.56 +/- 0.13 mA. The perfusion of 0.36 mM palmitate bound to 0.12 mM FABP lowered the VFT from 2.05 +/- to 0.19 mA to 1.47 +/- 0.23 mA, but 0.12 mM FABP alone did not affect the VFT. Perfusion with 0.36 mM palmitate bound to 0.12 mM FABP caused the VFT to fall more than perfusion with 0.36 mM palmitate bound to 0.12 mM albumin. Then the effects of verapamil perfusion or a low concentration of perfusate Ca2+ on VFT were examined. VFT was determined by electrical stimulation. Palmitate (0.6 mM) bound to 0.12 mM albumin lowered VFT. Verapamil 10(-7) M perfusion and a low concentration of Ca2+ (Ca2+ 1.67 mM) suppressed the FFA-induced fall of VFT. These results suggested that the arrhythmogenic action of FFA was related to Ca2+ overload in myocardial cells.

To test the hypothesis that structural abnormalities exist in the kidney membrane of spontaneously hypertensive rats, we examined the effect of long-term administration of coenzyme Q10 on membrane lipid alterations in the kidney of stroke-prone spontaneously hypertensive rats (SHRSP). As compared with normotensive Wistar-Kyoto rats, renal membrane phospholipids, especially phosphatidylcholine and phosphatidylethanolamine, decreased and renal phospholipase A2 activity was enhanced with age in untreated SHRSP. Treatment with coenzyme Q10 attenuated the elevation of blood pressure, the membranous phospholipid degradation, and the enhanced phospholipase A2 activity. These results suggest that one factor contributing to the progress of hypertension is a structural membrane abnormality that alters the physical and functional properties of the cell membrane, and coenzyme Q10 might protect the renal membrane from damage due to hypertension in SHRSP.

The effect of NCO-700, a cystine protease inhibitor, on platelet-activating factor-induced platelet aggregation was determined. A newly synthesized cystine protease inhibitor (calcium-activated neutral protease and cathepsin B inhibitor), NCO-700 (bis[ethyl (2R, 3R)-3-[(S)-methyl-1-[4-(2,3,4-trimethoxyphenylmethyl) piperazine-1-ylcarbonyl]butylcarbonyl]oxiran-2-carboxylate]sulfate), inhibited platelet-activating factor-induced platelet aggregation. The inhibition was dependent on the preincubation time with NCO-700 and on the concentration of the inhibitor. The release of serotonin was also inhibited almost completely by the 20-min preincubation with 10−4 m NCO-700. Leupeptin also inhibited platelet-activating facor-induced platelet aggregation. But calcium-activated neutral protease inhibitor did not inhibit it. These observations suggest that NCO-700-sensitive protease(s) such as cystine protease may contribute to platelet aggregation induced by platelet-activating factor.

We studied the effects of endothelin on angiotensin converting enzyme (ACE) in cultured pulmonary artery endothelial cells. ACE activity was increased 2.5-fold by the addition of 1 x 10(-8) mol/l endothelin-1. Endothelin-1 also stimulated calcium influx and phospholipase C activity in a dose-dependent manner. Calcium influx, phospholipase C and ACE activity were suppressed 60-70% in the presence of endothelin-1 (10(-10) to 10(-6) mol/l) by 50 microliters neomycin. These results suggest that ACE was stimulated by endothelin-1 and that its activity may be closely related to phosphatidylinositol turnover stimulated by endothelin-1.

Atrial natriuretic peptide (ANP), a cardiac hormone, is known to be located in the atrial specific granules, but its presence and localization in the ventricular muscle of the human heart has not been examined fully. Using a specific antibody to human ANP, we studied the conduction system and ventricular muscle with immunohistochemical and ultrastructural methods in 30 hearts obtained at autopsy. These included 12 normal and 18 diseased hearts. In the normal hearts, ANP-positive granules, which were regularly observed in the atrial myocytes, were found in small quantities in the cells of the penetrating and branching bundles in 4 of 12, and in the cells of the ventricular free walls in 2 of the 12 hearts. In the diseased hearts, the positivity increased significantly (P less than 0.05), being found in 13 of 18 (72.2%) conduction systems and 10 of 18 (55.6%) ventricular muscles. The granules were confirmed to be immunoreactive with ANP by ultrastructural examination. Furthermore, the presence of ANP mRNA in the conduction system as well as in the ventricular myocytes was demonstrated by Northern blot hybridization for which we used the complementary DNA of human ANP. Thus, a small quantity of ANP appears to be synthesized and stored in the conduction system and ventricles of some normal hearts. However, ANP was shown to be present in a larger percentage of the diseased hearts.

In this study, we examined the effects of streptokinase on arachidonic acid release and prostacyclin biosynthesis in cultured bovine pulmonary artery endothelial cells. When intact cells were incubated with streptokinase, a significant stimulatory effect on prostacyclin biosynthetic activity in cells was evident without any cellular damage at all concentrations used (1–10 000 units/ml). Streptokinase also caused a marked release of arachidonic acid. It induced rapid phospholipid hydrolysis, resulting in the release of up to 15% of incorporated [ 3 H]arachidonic acid into the medium. After the addition of streptokinase, degradation of phosphatidylcholine and phosphatidylethanolamine was observed and lysophosphatidylcholine and lysophosphatidylethanolamine were produced. We also observed a transient rise in diacyglycerol after the addition of streptokinase. To test for phospholipase C activity, the release of incorporated [ 3 H]choline, [ 3 H]inositol and [ 3 H]ethanolamine into the culture medium was determined. The level of radioactive inositol showed an increase, but the changes in choline and ethanolamine were comparatively small. An increase in inositol was detectable within 1 min after streptokinase addition and peaked after 15 min. Inositol phosphate and inositol trisphosphate were released, and these releases were suppressed by the addition of neomycin (50 μM). These results suggest that streptokinase stimulates phospholipase A 2 and C activity, and that prostacyclin biosynthesis is subsequently increased in cultured endothelial cells.

1) Captopril was orally administered in a dose of 12.5 mg to 12 patients with congestive heart failure to follow changes in its blood concentration and determine changes in clinical test values. 2) The blood concentration of captopril reached its peak in 2 h after medication, the mean value being 274 ng/ml and the half-life 3.16 h. The Tmax and T1/2 were found to be extended as compared with those of normal humans and hypertensive patients that had been reported. No significant differences were noted between Group I of mild cases and Group II of serious cases. 3) Following administration of captopril, a rise in angiotensin I and renin activity and a reduction in aldosterone were noted. These were found to be correlated or inversely correlated with the changes in the blood concentration of captopril. Greater changes were noted in Group II than in Group I. All clinical test values in each group tended to return to the control value 6h after the administration.

In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wistar-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP2) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity (PI-PLC) was predominantly located in the cytosol, and its activity was similar in both strains. Membrane-bound PIP2-hydrolyzing PLC activity (PIP2-PLC) was significantly lower in 20-week-old SHR than in WKY, but there was no significant difference in soluble PIP2-PLC. Protein kinase C activity was significantly elevated in 20-week-old SHR and Ca2(+)-phospholipid-dependent phosphorylation was observed in the proteins of molecular weight 26, 32, 43, and 95 KDa. In 4-week-old prehypertensive SHR, there were no significant differences in PI-PLC, PIP2-PLC, or protein kinase C activities as compared with age-matched WKY. These data demonstrated that protein kinase C and membrane-bound PIP2-PLC are altered during the period of hypertension development. These alterations may have important roles in the development or maintenance of hypertensive cardiac hypertrophy in SHR.

To characterize the viscoelastic properties of the human aorta in vivo, the pressure-diameter relation was determined in the abdominal aorta in 15 subjects. Diameter was measured noninvasively with a highly sensitive ultrasonic displacement meter, while intra-aortic pressure was measured with a catheter tip micromanometer inserted from the femoral artery. The frequency-dependent changes in the pressure-strain modulus (Ep) of the aorta and the phase lag of diameter to pressure were calculated by frequency analysis of these wave forms at the mean blood pressure of 109 mm Hg. The Ep and the phase lag at the fundamental frequency component (1.2 +/- 0.3 Hz, mean +/- SD) were (1.52 +/- 0.57) x 10(6) dyne/cm2 and -6.7 degrees +/- 2.1 degrees, respectively. Although the phase lag at the fundamental frequency was in good agreement with the published in vitro data, the calculated phase lags above the second harmonic were inconsistent, which was probably due to the nonlinearity in the pressure-diameter relation. To separate the effect of this nonlinearity, analysis was conducted with a model consisting of a static nonlinear component representing the elasticity and a dynamic linear component representing the viscosity. This method of analysis revealed that the phase lag due to the viscous component provided relatively flat frequency response to the 10th harmonic. It was confirmed that the aortic wall viscosity showed no apparent difference between the in vivo and the in vitro experimental conditions.

In order to clarify the incidence of unstable angina (UA), the rate of mutual transition to other forms of ischemic heart disease, and the short term prognosis of asymptomatic patients, we followed prospectively 421 medically treated patients after through assessment of coronary artery disease for an average of 43 months. Three hundred and two episodes of UA had occurred in 139 patients. Of these 14 patients experienced major cardiac events. Sixteen patients had sudden cardiac events with no prior UA. Two hundred and sixty-six patients had no episodes of UA nor cardiac events; of these, 97 patients were followed for more than 49 months. Those with both effort and rest UA had a much more higher incidence of unstable episodes and a higher average age than those with effort UA alone and those with rest UA alone. The average transition rate to the cardiac events by month of patients with UA, by markovian analysis, was higher than those of the asymptomatic or stable angina patient groups. When background factors are taken account, these transition rates might help the physicians in determining the treatment of patients with ischemic heart disease at a specific stage of state.