Investigation of in vitro parameters and fertility of mouse ovary after storage at an optimal temperature and duration for transportation.

STUDY QUESTION: How do the temperature and duration of storage affect ovaries during transportation? SUMMARY ANSWER: Fertility is reduced with the extension of the storage duration. WHAT IS KNOWN ALREADY: Live birth has been reported after ovarian transport overnight on ice before freezing ovarian tissue, but there have been no basic investigations of ovarian storage conditions focused on fertility. There are no guidelines on optimal ovarian storage conditions and the maximum storage time during transportation. STUDY DESIGN, SIZE AND DURATION: Experiments were performed using C57BL/6J mice. Ovaries of 4-week-old mice were harvested, stored at 4, 14, 37 °C or room temperature (RT) for 24 h, and subjected to histological examination. Next, ovaries were stored at 4 °C for 4, 8 or 24 h and subjected to histological examination. Then orthotopic transplantation of ovaries, stored at 4 °C for 4, 8 or 24 h, was performed in 6-week-old C57BL/6J mice, and fertility was assessed by in vitro fertilization and embryo transfer. Freshly harvested ovaries were used as controls for comparison with ovaries stored under the above-mentioned conditions and experiments were repeated at least three times. PARTICIPANTS/MATERIALS, SETTING AND METHODS: In experiments on the ovarian storage temperature, haematoxylin-eosin (HE) staining was performed for histological examination. In experiments on the storage duration, HE staining, the terminal deoxynucleotidyl transferase dUTP nick end labelling assay, Ki-67 staining and electron microscopy were performed, and the numbers of follicles were counted. Fertility was assessed from the number of oocytes, and the rates of fertilization, embryo development, implantation and live birth. MAIN RESULTS AND THE ROLE OF CHANCE: Histological changes were minimal after storage of ovaries at 4 °C for up to 24 h. At 4 °C, there were no significant changes in the number of MII oocytes, fertilization rate or blastocyst development rate with storage up to 24 h. The implantation rate was 82.7 ± 17.3% in the control group, while it was 82.2 ± 7.7, 14.6 ± 14.6 and 4.4 ± 4.4% after storage for 4, 8 or 24 h, respectively. After 8 or 24 h of storage, the implantation rate was significantly lower in than in the control group (P< 0.05). The rate of live pups was 24.8 ± 13.2% in the control group, while it was 23.9 ± 6.6, 4.2 ± 4.2 and 4.4 ± 4.4% after storage for 4, 8 or 24 h, respectively. After 8 or 24 h of storage, the rate of live pups was significantly lower than in the control group (P< 0.05). LIMITATIONS, REASONS FOR CAUTION: Further investigations are needed in mammals with ovaries of a similar size to human ovaries, and should include the assessment of fertility following transplantation of frozen and thawed ovaries. WIDER IMPLICATION OF THE FINDINGS: The present results suggest that prolonging the ovarian storage time reduces fertility in mice. Thus, ovaries should be frozen immediately after harvesting or transported as rapidly as possible to minimize damage. To allow young cancer patients to preserve fertility, regional medical centres need adequate ovarian tissue cryopreservation techniques. STUDY FUNDING/COMPETING INTERESTS: This study supported by Department of Obstetrics and Gynecology, St. Marianna University School of Medicine. The authors have no competing interests to declare.

Hypertrophic cardiomyopathy with midventricular obstruction and apical aneurysm: a case report.

A 71-year-old woman had hypertrophic cardiomyopathy associated with midventricular obstruction and an apical aneurysm in the left ventricle. She had had abnormal electrocardiograms for more than 30 years and for the past year had been suffering from occasional attacks of dizziness and low systemic blood pressure. Holter 24-h electrocardiographic monitoring revealed ventricular paroxysmal contractions (676/day) with nonsustained ventricular tachycardia. Doppler echocardiography revealed paradoxical jet flow from the apical aneurysm to the left ventricular outflow during early diastole. Magnetic resonance imaging depicted midventricular hypertrophy and a dyskinetic thin apical wall, which were confirmed by angiography. Coronary angiograms showed no narrowing of the major extramural coronary arteries, but there was compression of aberrant coronary arteries apparently feeding the hypertrophic portion of the left ventricular wall. Stress thallium-201 myocardial imaging showed a persistent severe defect in the left ventricular apex. A hemodynamic study revealed low cardiac output and an intraventricular pressure gradient (approximately 90 mmHg) between the left ventricular apical high-pressure chamber and the subaortic low-pressure chamber. The present case represents a rare combination of hypertrophic cardiomyopathy, midventricular obstruction, and an apical aneurysm in an elderly woman. Myocardial ischemia may have played an important role in the genesis of the apical aneurysm.

Left ventricular relaxation abnormality is detectable by analysis of the relaxation time constant in patients with atrial fibrillation.

Left ventricular (LV) contractility is constantly changing during atrial fibrillation (AF), which is dependent on the force-interval relationships. However, no information has been available on LV relaxation in patients with both AF and impaired LV systolic function. LV pressure was measured with a catheter-tipped micromanometer and the time constant of isovolumic LV pressure decline (tau(bf)) was calculated with best exponential fitting from more than 10 consecutive beats. Patients with AF (5 with mitral valvular disease, 6 with idiopathic dilated cardiomyopathy, and 1 with no underlying disease) were subdivided into 2 groups: group A, with ejection fraction (EF) <0.5 (n=7); and group B, with EF > or =0.5 (n=5). Linear correlation coefficients (r) between tau and RR2, RR2/RR1, LV peak systolic pressure (peak LVP) were calculated. Although tau did not show a discrepancy between the 2 groups, tau(bf) correlated better with RR2/RR1 only in the group A patients. The relation between tau and peak LVP showed a good correlation with a steep slope (R, Deltatau/Deltapeak LVP) only in the group A patients (accentuated afterload-dependence). R was significantly different between the 2 groups. Thus, a beat-to-beat analysis of tau may be a practical and feasible way for detecting LV relaxation abnormality in patients with AF.

Cloning of hypoxia-inducible factor 1alpha cDNA from chick embryonic ventricular myocytes.

Hypoxia-inducible factor 1 (HIF-1) is composed of HIF-1alpha and arylhydrocarbon nuclear receptor translocator (ARNT), which belong to the basic-helix-loop-helix-Per/ARNT/Sim (bHLH-PAS) family of transcription factors. HIF plays key roles in oxygen homeostasis and embryonic cardiovascular development. In this study, we have cloned cDNAs encoding the chick HIF-1alpha from cultured embryonic ventricular myocytes (CEVM) and then examined its expression in various embryonic tissues. The deduced amino acid sequence of the chick HIF-1alpha cDNA showed 79% identity with that of the human HIF-1alpha cDNA. In contrast, sequence homology between the chick HIF-1alpha and endothelial PAS protein 1 (EPAS1), another member of the bHLH-PAS proteins, was only low (49%). HIF-1alpha mRNA was expressed abundantly in CEVM, but scarcely in the liver, which was quite different from expression pattern of EPAS1 mRNA. These data suggest that HIF-1alpha may be involved in embryonic cardiovascular development. HIF-1alpha and EPAS1 may play distinct roles during developmental stages.

Ets-1 is involved in transcriptional regulation of the chick inducible nitric oxide synthase gene in embryonic ventricular myocytes.

In order to elucidate roles of Ets family of transcription factors in transcriptional activation of inducible nitric oxide synthase (iNOS) genes, we analyzed the chick iNOS gene expression in cultured chick embryonic ventricular myocytes (CEVM). Deletional analysis and site-directed mutagenesis demonstrated that both the Ets/PEA3 site (-221 to -216 bp) and the kappaB site (-101 to -93 bp) of the 5'-flanking region of the chick iNOS gene were involved in the maximal activation of the lipopolysaccharide (LPS)-induced expression of the reporter (luciferase) gene, although the proximal kappaB site played the more essential role. Electrophoretic mobility shift assay revealed that LPS augmented the nuclear protein bindings to the Ets/PEA3 as well as kappaB motifs. Ets-1, one of the Ets proteins, was suggested to be bound to the Ets/PEA3 oligonucleotide. By Northern blot analysis, LPS was shown to induce iNOS mRNA in CEVM, along with a preceding increase in the levels of c-ets-1 mRNA. Ets-1 may be involved in the iNOS gene transcription in CEVM, presumably through interacting with the NF-kappaB.

Expression of genes encoding vascular endothelial growth factor and its Flk-1 receptor in the chick embryonic heart.

Vascular endothelial growth factor (VEGF) is known to play an essential role in embryonic vascular development. The heart is one of the main organs that produce VEGF, but it is still unknown how expression of VEGF gene is regulated in embryonic cardiac myocytes. Thus, we cloned cDNAs encoding VEGF and its receptor (a KDR/flk-1 or Quek1 homologue) from cultured 10-day-old chick embryonic ventricular myocytes (CEVM). Reverse transcription-polymerase chain reaction revealed that the chick VEGF mRNAs consisted of at least four different species corresponding to the isoforms of 190, 166, 146 and 122 amino acids. In the embryonic heart and CEVM, the isoforms of 166 and 122 amino acids were dominant. Northern blot analysis detected an abundance of VEGF mRNA in both the embryonic heart and CEVM, even at the basal state. The levels of VEGF mRNA in CEVM were significantly augmented by forskolin (100 microM), or phorbol 12-myristate, 13-acetate (200 nM) in a time-dependent manner in CEVM. In contrast, the basal levels of VEGF mRNA were attenuated by genistein (100 microM), but not by H89 (100 microM) or bisindolylmaleimide (75 microM). Northern blot analysis also detected the chick flk-1 mRNA in abundance in the embryonic heart, and to a much lesser extent in CEVM. The expression levels of VEGF and flk-1 mRNA species were continuously high in the 6, 8 and 10-day-old chick embryonic hearts. In the 10-day-old embryonic hearts, in situ hybridization confirmed that mRNA encoding VEGF was mainly expressed in ventricular myocytes. In contrast, the flk-1 mRNA was detected in the microvascular endothelial cells, and to a lesser extent in the ventricular myocytes. These data suggest that VEGF is produced in embryonic ventricular myocytes, even at the basal state, and that the levels of VEGF mRNA may be differently regulated by various protein kinases. VEGF produced by the chick embryonic heart may play important roles in embryonic cardiovascular development by acting on surrounding endothelial cells and, possibly, on ventricular myocytes themselves.

Hemodynamic and thermal responses to a 30-minute constant-workload aerobic exercise in middle- or old-aged patients with cardiovascular diseases.

The aim of the present investigation was to compare the hemodynamic and thermal responses to a 30-min aerobic exercise between middle- or old-aged patients with normal left ventricular function and those with left ventricular dysfunction. Constant-load sitting ergometer exercise of approximately 90% of the subject's oxygen uptake (VO2) at the anaerobic threshold for 30 min was conducted in 21 patients with left ventricular dysfunction (61+/-10 years, left ventricular ejection fraction (LVEF) 35+/-7%) and 24 patients with normal left ventricular function (59+/-9 years, LVEF 71+/-7%). Heart rate (HR), blood pressure, deep temperatures in the forehead and thigh, and forearm skin blood flow (SkBF) were measured every minute, and cardiac output (CO) and stroke volume (SV) were determined every 10 min with the dye-dilution technique during the exercise. Patients of both groups exhibited a progressive elevation in each temperature and an increase in SkBF during the exercise. Although the VO2 and CO remained stable, almost the same magnitude of decrease in SV as increase in HR was seen after the 10th min of exercise in both groups. The magnitude of the decrease in SV was greater in old-aged than middle-aged patients with left ventricular dysfunction. Thus, the downward drift in SV during a 30-min constant-load aerobic exercise might not be influenced by left ventricular function, but intensified by aging in patients with left ventricular dysfunction.

Low-dose dobutamine radionuclide ventriculography for prediction of myocardial viability: quantitative analysis of regional left ventricular function.

BACKGROUND: It is important to distinguish viable myocardium from necrotic tissue in order to decide upon therapy in patients with ischemic heart disease. HYPOTHESIS: We verified the hypothesis that quantitative analysis of regional left ventricular function using low-dose dobutamine radionuclide ventriculography (RNV) can sensitively predict myocardial viability and compared its usefulness with thallium-201 (201Tl) single-photon emission computed tomography (201Tl-SPECT). METHODS: Radionuclide ventriculography at rest and during low-dose dobutamine infusion (5 micrograms/kg/min), 201Tl-SPECT, and coronary angiography were performed in 51 subjects with severe ischemia-related stenosis of coronary arteries and 3 subjects without coronary artery disease. 201Tl uptake was assessed as normal (control), low perfusion (LP), or defect. We compared the response of regional function to dobutamine with the regional 201Tl uptake. The accuracy of both methods for identifying viable myocardium was investigated in 17 patients who underwent successful coronary revascularization, with a resulting improvement in wall motion. RESULTS: The increase in regional ejection fraction (delta r-EF) in response to dobutamine was significantly greater in the control (12 +/- 6%) and LP (16 +/- 11%) regions than in the defect (5 +/- 10%) regions. The increase in one-third regional ejection fraction (delta r-1/3EF) was also significantly higher in the control (14 +/- 7%) and LP (10 +/- 8%) regions than in the defect regions (5 +/- 6%). We defined myocardial viability as a delta r-EF > 5% or a delta r-1/3EF > 2%. The sensitivity and specificity of the delta r-EF for identification of myocardial viability were 91.4 and 55.5%, respectively. The sensitivity and specificity of the delta r-1/3EF were 91.4 and 66.6%, respectively; the corresponding values for 201Tl SPECT were 74.2 and 77.8%. CONCLUSION: Low-dose dobutamine RNV with quantitative analysis of regional left ventricular function was more sensitive for identification of viable myocardium than 201Tl-SPECT.

High-resolution imaging of coronary calcifications by intense low-energy fluoroscopic X-ray obtained from synchrotron radiation.

PURPOSE: To obtain an intense monochromatic low-energy X-ray from synchrotron radiation (SR) and apply it to detect coronary calcifications. METHODS AND RESULTS: The SR beam was reflected with a silicon crystal to be expanded (150 mm in height and 80 mm in width) and to be monochromatized at an energy level of 37 keV. The X-ray was intermittently irradiated to obtain dynamic imaging of 30 images/s. Images were recorded by a digital fluorography system. The low-energy X-ray from SR sharply visualized calcification of coronary arteries, while conventional X-ray could not visualize coronary calcification. CONCLUSION: The intense monochromatic low-energy X-ray from SR is sensitive, has high-resolution for imaging coronary calcification and may serve as a screening method for coronary artery disease.

Lipopolysaccharide augments expression and secretion of vascular endothelial growth factor in rat ventricular myocytes.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is highly expressed in the myocardium under various stimuli including hypoxia and ischemia. On the other hand, lipopolysaccharide (LPS) causes systemic inflammatory response syndrome (SIRS), which consists of systemic pathophysiological changes related to vascular hyperpermeability. To test the hypothesis that VEGF is one of the important mediators of SIRS, we examined effects of LPS on the VEGF expression and secretion in cultured neonatal rat ventricular myocytes. LPS (10 microg/ml) rapidly (within 1 h) augmented the levels of VEGF mRNA in these cells. Pharmacological inhibition of nucleic factor-kappaB or tyrosine kinases did not affect the LPS-induced augmentation of VEGF mRNA expression, while these treatments markedly suppressed the up-regulation of inducible nitric oxide synthase (iNOS) expression by LPS. The VEGF concentrations in the conditioned media were also significantly increased by the LPS treatment of 6 h. In conclusion, LPS augments VEGF expression and secretion in rat ventricular myocytes, suggesting that VEGF may be involved in pathogenesis of SIRS. LPS may induce VEGF mRNA through the signaling pathways that are distinct from those responsible for the iNOS induction.

An adult case with an abnormal right ventricular structure causing intraventricular pressure gradient and a history of aphthous stomatitis and thrombophlebitis.

We report a 50-year-old man with a right ventricular structure causing an intraventricular pressure gradient. He had been diagnosed as vasculo-Behçet with a history of aphthous stomatitis and thrombophlebitis. He had also been suffering from atrial flutter and mild right-side heart failure. Echocardiography showed that there was an abnormal structure attached to the right ventricular free wall and protruding into the cavity, and that it caused the pressure gradient estimated to be approximately 19 mmHg. Chest X-ray computed tomography demonstrated that the structure was partially calcified. Magnetic resonance imaging depicted the structure separating the right ventricle into two chambers. Angiographic study revealed a markedly enlarged right atrium and a filling defect at the mid-portion of the right ventricle, which divided the right ventricular cavity into two parts. Hemodynamic study showed a slightly elevated right atrial pressure (mean 7 mmHg) and a peak-to-peak intraventricular pressure difference of 18 mmHg in the right ventricle. The diastolic pressure tracing of the right ventricular low pressure chamber showed a 'dip and plateau' pattern. Although the pathological features of the abnormal right ventricular structure in this case were not fully clarified, abnormal muscle bundle and/or endocardial fibrosis, which were reported to be associated with Behçet's disease, may have contributed to its generation.

Biological reactions in the heart: what should we do in 'cardiology'?

Humankind is on a similar evolutionary process to animals. Biological reactions in the human heart will be reviewed, and consideration will be made about what can be done in cardiology, from the viewpoints of basic, clinical and community medicine. Functional reactions of the heart to acute loading (exercise, etc) comprise myocardial contractility, preload, pump function and peripheral factors, and are mobilized step by step in that order, to maintain normal functioning. Morphological reactions to chronic loading (hypertension etc) comprise hypertrophy and dilatation, which are caused by mechanical and nonmechanical factors, but may not always be mobilized to maintain normal functioning. Various neurohumoral factors take part in the mechanisms, and modifications, of these reactions. They act in a complex manner according to the biological conditions, and may not always act to maintain normal functioning. The biological reactions in the heart (ie, Basic Cardiology) should not be interpreted as having purpose; that is, putting a value on humankind, although medical treatment (Clinical Cardiology) and the solution of health problems in the community (Community Cardiology) should be done from this viewpoint.

Platelet-derived growth factor induces cellular growth in cultured chick ventricular myocytes.

OBJECTIVES: Platelet-derived growth factor (PDGF) stimulates growth in various types of cells, but little is known about its effect on cardiac myocytes. Therefore, we examined whether PDGF had a direct effect on cardiac myocytes and investigated their intracellular signaling pathways. METHODS: A primary culture of chick embryonic (Hamburger and Hamilton stage 36) ventricular myocytes was prepared. Cellular growth was estimated by 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide assay and 5-bromo-2'-deoxyuridine incorporation assay. The number of PDGF binding sites was measured by binding assay. Induction of c-fos mRNA was analyzed by Northern blot analysis. The binding activity of activator protein (AP)-1 was examined by electrophoretic mobility shift assay. The activation of mitogen-activated protein kinase (MAPK) and signal transducers and activators of transcription (STATs) was analyzed by Western blot analysis, immunoprecipitation, and immunocytochemistry. Furthermore, intracellular Ca2+ concentration ([Ca2+]i) was measured with indo-1 and L-type Ca(2+)- channel current (ICa) was recorded with the patch clamp technique. RESULTS: PDGF-AB and -BB, but not PDGF-AA, increased viable cell number (5 ng/ml of PDGF-AA, -AB, -BB: 101 +/- 4%, 115* +/- 4%, 122* +/- 4%, respectively, n = 4, *P < 0.05) and DNA synthesis (104 +/- 11%, 202* +/- 18%, 295* +/- 25%, respectively, n = 4, *P < 0.05). Scatchard analysis demonstrated that the maximal number of PDGF-AA, -AB, -BB binding sites was 5 +/- 1, 63 +/- 12, 126 +/- 24 fmol/10(6) cells, respectively. PDGF-BB provoked induction of c-fos mRNA and increases in binding activity to the AP-1 site. PDGF-BB also induced tyrosine phosphorylation and nuclear translocation of MAPK. The c-fos induction, the increased AP-1 binding activity and the acceleration of DNA synthesis were all attenuated by genistein (100 microM) or MAPK kinase inhibitor (10 or 50 microM PD98059). Interestingly, protein kinase C inhibitor (250 nM calphostin C) attenuated the increases of AP-1 binding activity to some extent, but did not inhibit the c-fos induction at all. The phosphorylation states of STATs were not significantly affected by PDGF-BB. PDGF-BB did not alter [Ca2+]i or ICa. CONCLUSIONS: We conclude that PDGF can exert direct effects on embryonic cardiac myocytes and induce their growth. MAPK cascade may play an important role in the PDGF-induced embryonic myocardial growth.

Dynamic intravenous coronary angiography using 2D monochromatic synchrotron radiation.

A method of examination for coronary artery disease that is less invasive and easier than coronary angiography (CAG) has been sought. We have developed a dynamic intravenous coronary angiography (IVCAG) system using synchrotron radiation (SR) and have used it clinically. Four patients suspected of having angina pectoris underwent IVCAG. An SR beam was reflected asymmetrically with a silicon crystal to produce a wide (150 mm x 80 mm) and monochromatic (37 keV) X-ray beam, with an energy level to achieve high sensitivity to the contrast agent. Following an intravenous injection of contrast agent, irradiation was applied for 4 ms periods at 33 ms intervals for dynamic IVCAG at 30 images s-1. Images were acquired with an image intensifier and recorded with a digital fluorography system. The dynamic images permitted clear visualization of the coronary arteries and permitted evaluation of coronary anatomy. Two patients exhibited no stenotic lesions, one patient had a 90% stenosis in the right coronary artery, and the remaining patient had a 25% stenosis at the site of previous percutaneous transluminal coronary angioplasty in the left anterior descending artery (LAD). The total irradiation doses used for IVCAG were less than those for conventional angiography. Dynamic IVCAG can be readily used for the evaluation of coronary arteries.

Role of endothelin in deterioration of heart failure due to cardiomyopathy in hamsters: increase in endothelin-1 production in the heart and beneficial effect of endothelin-A receptor antagonist on survival and cardiac function.

BACKGROUND: We previously reported that chronic endothelin (ET) receptor blockade ameliorated the survival rate and cardiac hemodynamics in rats with chronic heart failure (CHF) due to myocardial infarction. However, it remains unclear whether ET-1 is involved in the pathophysiology of cardiomyopathy, which is one of the major causes of CHF. Accordingly, we investigated the production of ET-1 in the heart and the effect of chronic ETA receptor blockade on survival rate and cardiac function in the Bio 14.6 hamster, which is an idiopathic model of CHF caused by cardiomyopathy. METHODS AND RESULTS: We used 52-week-old Bio 14.6 cardiomyopathic hamsters and age-matched F1b normal hamsters. The expression of preproET-1 mRNA and the ET-1 level in the hearts were markedly higher in the cardiomyopathic hamsters than in the normal hamsters. The cardiomyopathic hamsters showed severe CHF, illustrated by lower left ventricular (LV) +dP/dt/Pmax and right ventricular (RV) +dP/dt/Pmax and by higher LV end-diastolic pressure (EDP), RVEDP, and central venous pressure compared with the normal hamsters. Long-term (9 weeks) treatment with an ETA antagonist (TA-0201, 1.3 mg. kg-1. d-1) markedly increased survival of cardiomyopathic hamsters (untreated, 16%; TA-0201-treated, 65.2%; P<0.001). After 6 weeks of treatment, LV +dP/dt/Pmax and RV +dP/dt/Pmax were significantly higher and LVEDP and RVEDP were lower in the TA-0201-treated group than in the untreated group, suggesting that chronic TA-0201 treatment effectively prevented deterioration of cardiac dysfunction. CONCLUSIONS: In the cardiomyopathic hamsters with CHF, the production of ET-1 in the heart was markedly increased, and chronic ETA receptor blockade greatly ameliorated survival and cardiac dysfunction. These results suggest that ET-1 plays an important role in the deterioration of CHF caused by cardiomyopathy, and ETA antagonists may exert therapeutic effects in CHF due to cardiomyopathy.

Expression of endothelin-1, ETA and ETB receptors, and ECE and distribution of endothelin-1 in failing rat heart.

Endothelin (ET)-1 has a positive inotropic effect and induces hypertrophy in cardiomyocytes. We previously reported that the peptide level of ET-1 is increased in the failing heart of rats with chronic heart failure (CHF) and that treatment with an ETA-receptor antagonist greatly improves survival in rats with CHF. However, precise analysis for alteration of the myocardial ET system in the failing heart is not known. In this study, we used rats with CHF due to chronic myocardial infarction. Sham-operated rats served as a control. The results showed that the level of preproendothelin (preproET)-1 mRNA and the peptide level of ET-1 were markedly increased in the heart of rats with CHF, whereas the expression of endothelin-converting enzyme (ECE)-1 mRNA in the heart did not differ between CHF and control rats. The intensity of ET-1 staining (ET-1-like immunoreactivity) in cardiomyocytes was markedly stronger in rats with CHF than in control rats, and the fibrotic tissues of the infarcted area were not stained. The mRNA and protein levels of both ETA and ETB receptors in the heart were significantly higher in rats with CHF than in control rats. The present study suggests that the increase in ET-1 peptide level in the heart of the rats with CHF originated from upregulation of preproET-1 mRNA, which was not attendant with the alteration of ECE-1 mRNA expression, and that both the ETA- and ETB-receptor systems are greatly accelerated in the failing heart.