The exact mechanisms responsible for the progression of heart failure remain unclear. We investigated the in vivo relationship between the incidence of apoptotic cell death and left ventricular function serially from the beginning of hypertension to decompensated heart failure in Dahl salt-sensitive rats. Dahl salt-resistant and Dahl salt-sensitive rats were fed on a high-salt diet from 6 weeks of age. Systolic blood pressure was recorded by the tail-cuff method every week. Cardiac function in vivo was evaluated by echocardiography and cardiac catheterization. Cardiomyocyte apoptosis was detected by the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) method. The gene expression of Bax, Bcl-2 and Bcl-xL was analysed by Northern blotting. The TUNEL method revealed that the incidence of cardiomyocyte apoptosis was significantly increased in the hearts of 18-week-old Dahl salt-sensitive rats (apoptotic index 1.3±0.1%). Northern blot analysis revealed that the Bcl-xL mRNA level increased gradually during the progression towards heart failure. In conclusion, these data suggest that cardiomyocyte apoptosis is a terminal event, and plays a role as an aggravating factor in the vicious cycle of heart failure.

Congestive heart failure is a common and serious complication in patients undergoing chronic dialysis. However, there have been no studies on preferential medical therapies to improve left ventricular function in haemodialysis patients. β-Blocker treatment is known to improve left ventricular function in patients with dilated cardiomyopathy; moreover, plasma levels of noradrenaline and natriuretic peptides are sensitive markers of left ventricular dysfunction. The present study investigated whether β-blocker treatment could improve left ventricular function in haemodialysis patients with a dilated left ventricle. Our study group comprised 14 haemodialysis patients with a dilated left ventricle, who had undergone maintenance haemodialysis for a mean of 11 years. The following haemodynamic parameters were evaluated before and after 4 months of treatment with the β-blocker metoprolol: left ventricular dimension at end-systole and end-diastole, and fractional shortening. Plasma levels of noradrenaline, atrial natriuretic peptide and brain natriuretic peptide were also determined. Dry body weight and haemoglobin concentration showed no significant change after compared with before treatment with metoprolol. Heart rate decreased significantly, from 79±9 beats/min to 69±9 beats/min, but systolic blood pressure remained unchanged. The left ventricular dimension both at end-systole and at end-diastole was decreased, and fractional shortening increased significantly. Plasma levels of noradrenaline did not change significantly, but those of atrial natriuretic peptide and brain natriuretic peptide decreased markedly [from 100±89 pg/ml to 46±29 pg/ml ( P = 0.0051) and from 549±516 pg/ml to 140±128 pg/ml ( P = 0.0035) respectively]. In conclusion, β-blocker therapy with metoprolol can markedly attenuate left ventricular remodelling and decrease the plasma levels of natriuretic peptides in haemodialysis patients with a dilated left ventricle.

GADD153 (growth arrest- and DNA damage-inducible gene 153) is expressed at very low levels in growing cells, but is markedly induced in response to a variety of cellular stresses, including glucose deprivation, exposure to genotoxic agents and other growth-arresting situations. Forced expression of GADD153 induces cell cycle arrest in many types of cells. It is also reported that GADD153 is directly associated with apoptosis. Recently we have reported that platelet-derived growth factor (PDGF)-BB induces apoptosis in cultured vascular smooth muscle cells (VSMC), but only when 100% confluency is reached. These results suggested that cell–cell contact inhibition (cell growth arrest) may be a critical factor for induction of VSMC apoptosis by PDGF-BB. In the present study, we explored the role of GADD153, one of a number of growth-arrest-related gene products, in the molecular mechanisms of VSMC apoptosis in vitro and in vivo . GADD153 was markedly induced at both the mRNA and protein levels, in parallel with the induction of VSMC apoptosis, after treatment with PDGF-BB. Moreover, overexpression of GADD153 in VSMC significantly reduced cell viability and induced apoptosis. In the carotid artery balloon injury model in rats, GADD153 protein was expressed in apoptotic VSMC which were positively stained by in situ DNA labelling. These results demonstrate an important role for GADD153 in the molecular mechanisms of VSMC apoptosis.

Various alterations in molecular and cellular events have been considered as possibly contributing to the cardiac remodelling that occurs during the transition from compensated hypertrophy to heart failure. The aim of the present study is to clarify (1) whether cardiac apoptosis occurs during the transition from compensated hypertrophy to decompensated heart failure, and (2) whether expression of the genes encoding Bax (an apoptosis inducer) and Bcl-xL and Bcl-2 (apoptosis inhibitors) is altered during this transition. We used 12-month-old and 20-month-old male spontaneously hypertensive rats (SHR 12 and SHR 20 respectively) and age-matched Wistar–Kyoto rats (WKY 12 and WKY 20 respectively). These rats were killed after measurement of haemodynamic parameters by transthoracic echocardiography and use of a tipmanometer via the right carotid artery. The expression of bcl-2 , bcl-xL and bax was analysed by Northern blotting. Samples were also fixed in 4% paraformaldehyde for in situ nick end-labelling (TUNEL) methods and immunohistochemistry. SHR 12 had well compensated left ventricular hypertrophy with normal fractional shortening and normal end-systolic wall stress. In contrast, the hearts of SHR 20 developed decompensated dilatation, with a decrease in fractional shortening and an increase in end-systolic wall stress. TUNEL-positive cells were seen exclusively in the hearts of SHR 20 . The major cell types that showed TUNEL-positive nuclei were non-cardiomyocytes. The expression of bax remained unchanged during the transition to heart failure. However, there was increased expression of bcl-xL in the failing stage, whereas the expression of bcl-2 remained unchanged. Immunohistochemical studies revealed that Bcl-xL protein was up-regulated in the hearts of SHR 20 . In conclusion, non-cardiomyocyte apoptosis may play a contributory role in the remodelling that occurs in the transition from compensatory hypertrophy to decompensated heart failure. In addition, it is suggested that enhanced expression of bcl-xL plays an important role in the preservation of cardiomyocytes during this transition.

We performed balloon injury in the rat carotid artery and identified intimal thickening after injury. Balloon-injured carotid arteries showed maximum thickness of the neointima on the 14th day before complete endothelial cell regeneration. In this lesion we identified apoptosis of vascular smooth muscle cells (VSMCs) by in situ DNA labelling and electron microscopy in the neointima on the 14th day after injury. mRNA expression levels of bcl-2 , bax , bcl-x , p53 and caspase-1 were determined by the reverse transcriptase–polymerase chain reaction method both in injured and uninjured carotid arteries. Neither bcl-2 nor bcl-xl mRNA expression was detected in either injured or uninjured arteries, whereas bax and p53 mRNA expression was identified and their mRNA levels were not altered after balloon injury. In contrast, both bcl-xs and caspase-1 mRNA was detected and was markedly induced only in the injured carotid artery. Positive staining for immunoreactive Bcl-x was observed specifically in the injured arterial wall and co-localized with positive staining of nuclei identified by in situ DNA labelling. We conclude that two opposite cellular responses, VSMC proliferation and apoptosis, exist together in the neointima of the rat carotid artery after balloon injury, and selective induction of Bcl-xs expression is a key regulator of VSMC apoptosis in the process of vascular remodelling.

This study was designed to clarify whether gene expression in the cardiac sarcoplasmic reticulum [sarcoplasmic reticulum Ca 2+ -ATPase (SERCA), phospholamban, ryanodine receptor and calsequestrin] changes in accordance with left ventricular functional alterations in the volume-overloaded heart. Further, the effect of the angiotensin II type 1 receptor antagonist, TCV-116, on the expression of these genes was also evaluated. Left ventricular fractional shortening was significantly increased at 7 days, had returned to control levels at 21 days, and had significantly decreased at 35 days after the shunt operation, compared with sham-operated rats. The level of SERCA mRNA was significantly decreased at both 21 days and 35 days after the shunt operation. The levels of ryanodine receptor and phospholamban mRNAs were significantly decreased at 35 days in shunt-operated rats. The decrease in the SERCA mRNA level preceded the development of cardiac dysfunction. The levels of SERCA and ryanodine receptor mRNAs were correlated positively with left ventricular fractional shortening ( r = 0.73, P < 0.0001 and r = 0.61, P < 0.01 respectively). Attenuation of the decrease in left ventricular fractional shortening occurred on treatment with TCV-116. After the treatment with TCV-116, the levels of SERCA and phospholamban mRNAs were restored to the respective values in sham-operated rats. Ryanodine receptor mRNA levels remained unchanged after treatment with TCV-116. These results indicate that the down-regulation of SERCA and ryanodine receptor mRNA levels may be related to cardiac dysfunction in the volume-overloaded heart. In addition, treatment with an angiotensin II receptor antagonist may restore the altered sarcoplasmic reticulum mRNA levels to control levels, and this may result in attenuation of the functional impairment in the volume-overloaded heart.

1. The aim of this study was to elucidate the pathophysiological role of adrenomedullin and the relation between adrenomedullin and other hormones in patients with hypertrophic cardiomyopathy. 2. Fourteen patients with hypertrophic obstructive cardiomyopathy (HOCM), 26 patients with hypertrophic non-obstructive cardiomyopathy (HNCM) and 14 normal control subjects participated in this study. Radioimmunoassay for plasma adrenomedullin concentration was performed with adrenomedullin-M antibody. Plasma levels of endothelin-1, atrial and brain natriuretic peptides and noradrenaline were also measured. 3. Plasma levels of adrenomedullin were higher in patients with hypertrophic cardiomyopathy (8.43 ± 3.73 pmol/l) than in normal controls (5.24 ± 0.44 pmol/l, P < 0.005). There was no significant difference between HOCM and HNCM patients. There was a weak correlation between plasma levels of adrenomedullin and total 12-lead QRS voltage in patients with hypertrophic cardiomyopathy ( r = 0323, P < 0.05) 4. Plasma levels of endothelin-1, atrial and brain natriuretic peptides were higher in hypertrophic cardiomyopathy than in normal controls. Endothelin-1 showed no significant difference between HOCM and HNCM patients, but atrial and brain natriuretic peptides were higher in HOCM than in HNCM patients. There was a positive correlation between plasma levels of adrenomedullin and endothelin-1 ( r = 0.575, P < 0.0001), but no correlation between plasma levels of adrenomedullin and atrial natriuretic peptide, brain natriuretic peptide and noradrenaline. 5. Our results indicate that adrenomedullin may play an important role to maintain haemodynamics in patients with hypertrophic cardiomyopathy, and its action may be related to endothelin-1 but independent of atrial natriuretic peptide, brain natriuretic peptide and noradrenaline.

1. We assessed the changes of atrial natriuretic peptide and brain natriuretic peptide gene expression associated with progression and regression of cardiac hypertrophy in renovascular hypertensive rats (RHR). 2. Two-kidney, one-clip hypertensive rats (6-week-old male Wistar) were made and studied 6 (RHR-1) and 10 weeks (RHR-2) after the procedure. Regression of cardiac hypertrophy was induced by nephrectomy at 6 weeks after constriction, and the nephrectomized rats were maintained further for 4 weeks (nephrectomized rat: NEP). Sham operation was performed, and the rats were studied after 6 (Sham-1) and 10 weeks (Sham-2). Atrial natriuretic peptide and brain natriuretic peptide gene expression in the left ventricle was analysed by Northern blotting. 3. Plasma atrial natriuretic peptide and brain natriuretic peptide were significantly higher in RHR-1 and RHR-2 than in Sham-1, Sham-2 and NEP. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels in RHR-1 were approximately 7.2-fold and 1.8-fold higher than those in Sham-1, respectively, and the corresponding levels in RHR-2 were 13.0-fold and 2.4-fold higher than those in Sham-2, respectively. Atrial natriuretic peptide and brain natriuretic peptide mRNA levels of NEP were normalized. Levels of atrial natriuretic peptide and brain natriuretic peptide mRNA were well correlated positively with left ventricular weight/body weight ratios. There was a significant positive correlation between the levels of atrial natriuretic peptide and brain natriuretic peptide mRNA ( r = 0.86, P <0.01). 4. We conclude that the expression of atrial natriuretic peptide and brain natriuretic peptide genes is regulated in accordance with the degree of myocardial hypertrophy and that the augmented expression of these two natriuretic peptides may play an important role in the maintenance of cardiovascular haemodynamics in renovascular hypertension.

1. This study was conducted to assess the role of atrial and brain natriuretic peptides during acute myocardial ischaemia associated with dynamic exercise. 2. Study subjects consisted of 35 angiographically proven patients with angina pectoris and 35 angiographically normal control subjects. All subjects underwent 201 Tl dynamic exercise testing. The presence and localization of the exercise-induced acute myocardial perfusion defect were assessed by 201 Tl single-photon emission computed tomography. The severity score was calculated using the early image for quantitative assessment of the acute myocardial perfusion defect. 3. Plasma levels of atrial natriuretic peptide increased from 21.3 ± 3.8 to 72.2 ± 26.7 pg/ml ( P < 0.01) in the angina pectoris group, and increased from 19.4 ± 2.4 to 36.4 ± 17.4 pg/ml ( P < 0.01) in the control group during dynamic exercise. Plasma levels of brain natriuretic peptide increased from 2.8 ± 0.8 to 6.9 ± 2.6 pg/ml ( P < 0.01) in the angina pectoris group, but did not change significantly in the control group (from 2.7 ± 0.7 to 2.9 ± 1.0 pg/ml) during dynamic exercise. At peak exercise, plasma levels of these natriuretic peptides in the angina pectoris group were significantly higher than those in the control group ( P < 0.01). 4. At peak exercise, there were correlations between the plasma level of atrial natriuretic peptide and heart rate in both the angina pectoris and control groups ( P < 0.01, r = 0.46; P < 0.01, r = 0.51, respectively), but no significant correlations between the plasma level of brain natriuretic peptide and heart rate in either group. The plasma levels of these peptides at peak exercise correlated well with the severity score in the angina pectoris group (atrial natriuretic peptide, r = 0.71, P < 0.01; brain natriuretic peptide, r = 0.69, P < 0.01). 5. The present study showed that plasma levels of atrial and brain natriuretic peptides significantly increased during acute myocardial ischaemia associated with dynamic exercise.

1. In order to clarify whether the myocardial dysfunction observed in diabetic-rat hearts is an intrinsic property of the myocytes or not, we investigated cardiac function and myocyte contractile function in diabetic rats 5 weeks after the injection of streptozotocin. 2. Maximal and minimal d P /d t and time constant of isovolumic pressure fall were measured using a micromanometer in diabetic and age-matched control rats. 3. Isolated myocytes were enzymically obtained from each rat heart and were stimulated at 1 Hz (37°C) in a buffer containing 1.5 mmol/l Ca 2+ . The images of myocyte contractions were recorded by a video system. Normalized maximal velocity of shortening (maximal velocity of cell shortening/resting cell length; s −1 ), normalized maximal velocity of relengthening (maximal velocity of cell relengthening/resting cell length; s −1 ) and extent of shortening [(twitch amplitude/resting cell length) × 100;%] were analysed by a digitalized computer as contractile functions of the myocyte. 4. The maximal and minimal d P /d t in diabetic rats (7876, 5341 mmHg/s) were significantly lower than those in control rats (9349, 7876 mmHg/s). The time constant of isovolumic pressure fall in diabetic rats (12.7 ms) was significantly greater than that in control rats (8.6 ms). Moreover, the normalized maximal velocity of shortening, normalized maximal velocity of relengthening and extent of shortening in myocytes from diabetic rats (1.40 s −1 , 1.38 s −1 , 9.5%) were significantly lower than those in myocytes from control rats (1.64 s −1 , 1.60 s −1 , 11.8%). 5. These results suggest that contractile impairment in this diabetic-rat heart model is mainly due to an intrinsic abnormality of the cardiac myocytes.

1. To determine whether a persistent release of creatine kinase from the myocardium occurs in patients with hypertrophic cardiomyopathy, the activities of serum creatine kinase MM isoforms were measured in 22 patients with hypertrophic cardiomyopathy and in 14 normal control subjects. 2. Serum creatine kinase MB activity was significantly higher in patients with hypertrophic cardiomyopathy (7.8 ± 3.8 i.u./l) than in normal control subjects (0.4 ± 0.8 i.u./l; P < 0.01). 3. Serum MMa, MMb and MMc activities in patients with hypertrophic cardiomyopathy were 19.4 ± 4.1%, 26.7 ± 2.5% and 33.5 ± 7.0% of the total creatine kinase MM activity, respectively. These values for each isoform were significantly different from those in normal control subjects (11.3 ± 3.0%, 21.5 ± 4.4% and 40.7 ± 7.0%, respectively). The MMa/MMc activity ratio was significantly higher in patients with hypertrophic cardiomyopathy (0.61 ± 0.25) than in normal control subjects (0.30 ± 0.10; P < 0.01). 4. Our results indicate that a small amount of the myocardial tissue isoform of creatine kinase MM (MMa) is constantly released in many patients with hypertrophic cardiomyopathy.

1. The effects of renin inhibitor ES-8891 on renin synthesis and its secretion by the kidney were investigated in normotensive sodium-depleted marmosets. We measured plasma renin activity, plasma immunoreactive renin concentration, plasma angiotensin II concentration and kidney renin mRNA content after oral administration of ES-8891 (60 mg day −1 kg −1 ) for 1 week. 2. The mean blood pressure was significantly decreased ( P < 0.01) on day 7 after oral administration of ES-8891. There was no significant change in heart rate during the administration. 3. Oral administration of ES-8891 for 1 week markedly decreased the plasma renin activity, the plasma immunoreactive renin concentration and the plasma angiotensin II concentration (to 18%, 41% and 24% of the corresponding control values; P < 0.05 for each, n = 5). 4. The kidney renin mRNA content in ES-8891-treated marmosets was significantly lower than that in normal controls (4.2 ± 3.5 versus 12.8 ± 5.5 pg/μg of total RNA, means ± sd , P < 0.05, n = 5). 5. Oral administration of the renin inhibitor ES-8891 for 1 week not only inhibited plasma renin activity but also decreased renin synthesis and its secretion by the kidney.

1. We separated inactive renin in human plasma into two types, adsorbed and non-adsorbed, by chromatography on a concanavalin A-Sepharose column. About 75% of fresh plasma inactive renin was adsorbed to the column, and the rest passed through it. Non-adsorbed and adsorbed inactive renins were partially purified. 2. Non-adsorbed inactive renin had a molecular weight of 48000 and an isoelectric point of 5.44. Adsorbed inactive renin had a molecular weight of 46000 and isoelectric points of 5.56 and 5.80. 3. After activation with trypsin, both activated inactive renins were similar with respect to molecular weight (45000), thermostability, K m value (0.56 μmol/l) and pH profile. But pI values of both activated inactive renins differed. 4. These results indicate that there exist in human plasma two different types of inactive renin which differ in carbohydrate composition.

1. Human angiotensin-converting enzyme from kidney, lung, small intestine, plasma and urine was found to be immunologically indistinguishable by double immunodiffusion and inhibition of enzyme activity with specific anti-(human kidney enzyme) antibody. 2. Partial inhibition of the enzyme activities from hog, dog and rat sera by the antibody was found, but no inhibition was observed in the enzymes from sheep and bovine sera. 3. Human angiotensin-converting enzyme was located in two types of cells by an immunofluorescent technique using the purified antibody and fluorescein-labelled anti-(rabbit immunoglobulin) antibody. These were the vascular endothelial cells of alveolar capillaries and of pulmonary interstitial small vessels, and the epithelial cells of proximal tubules in the kidney and of mucous membrane in the small intestine.

1. The effects of prostaglandin (PG) E 1 , PGE 2 and PGF 2α on plasma renin substrate concentration in rats and renin substrate production by perfused rat liver were investigated. 2. After bilateral nephrectomy, both plasma renin substrate concentration and renin substrate production were remarkably elevated. 3. The increase in plasma renin substrate level and production of renin substrate by the liver after nephrectomy was markedly suppressed by the treatment with PGE 1 or PGE 2 . 4. In contrast PGF 2α had no suppressive effect on renin substrate synthesis. 5. These findings suggest that PGE 1 and PGE 2 might be inhibitory factors in the regulation of renin substrate synthesis.