Depressed contractile reserve and impaired calcium handling of cardiac myocytes from chronically unloaded hearts are ameliorated with the administration of physiological treatment dose of T3 in rats.

AIM: Chronic cardiac unloading causes a time-dependent upregulation of phospholamban (PLB) and depression of myocyte contractility in normal rat hearts. As thyroid hormone is known to decrease PLB expression, we examined whether thyroid hormone restores the depressed contractile performance of myocytes from chronically unloaded hearts. METHODS: Cardiac unloading was induced by heterotopic heart transplantation in isogenic rats for 5 weeks. Animals were treated with either vehicle or physiological treatment dose of 3,5,3'-triiodo-L-thyronine (T3) that does not cause hyperthyroidism for the last 3 weeks (n=20 each). RESULTS: In vehicle-treated animals, myocyte relaxation and [Ca2+]i decay were slower in unloaded hearts than in recipient hearts. Myocyte shortening in response to high [Ca2+]o was also depressed with impaired augmentation of peak-systolic [Ca2+]i in unloaded hearts compared with recipient hearts. In vehicle-treated rats, protein levels of PLB were increased by 136% and the phosphorylation level of PLB at Ser16 were decreased by 32% in unloaded hearts compared with recipient hearts. By contrast, in the T3-treated animals, the slower relaxation, delayed [Ca2+]i decay, and depressed contractile reserve in myocytes from unloaded hearts were all returned to normal levels. Furthermore, in the T3-treated animals, there was no difference either in the PLB protein level or in its Ser16-phosphorylation level between unloaded and recipient hearts. CONCLUSION: These results suggest that the treatment with physiological treatment dose of thyroid hormone rescues the impaired myocyte relaxation and depressed contractile reserve at least partially through the restoration of PLB protein levels and its phosphorylation state in chronically unloaded hearts.

Feasibility of rapid-sequence 31P magnetic resonance spectroscopy in cardiac patients.

PURPOSE: To determine the clinical feasibility of rapid-sequence phosphorus-31 magnetic resonance spectroscopy (31P-MRS) of the heart with cardiac patients using a 1.5T clinical MR system. MATERIAL AND METHODS: Twenty cardiac patients, i.e. dilated cardiomyopathy (DCM) 13 cases, hypertrophic cardiomyopathy (HCM) 3 cases, hypertensive heart diseases (HHD) 3 cases, and aortic regurgitation (AR) 1 case were examined using rapid cardiac 31P-MRS. Complete three-dimensional localization was performed using a two-dimensional phosphorus chemical-shift imaging sequence in combination with 30-mm axial slice-selective excitation. The rapid-sequence 31P-MRS procedure was phase encoded in arrays of 8 x 8 steps with an average of 4 acquisitions. The total examination time, including proton imaging and shimming, for the rapid cardiac 31P-MRS procedure, ranged from 10 to 15 min, depending on the heart rate. Student's t test was used to compare creatine phosphate (PCr)/adenosine triphosphate (ATP) ratios from the cardiac patients with those of the control subjects (n = 13). RESULTS: The myocardial PCr/ATP ratio obtained by rapid 31P-MRS was significantly lower (P < 0.001) in DCM patients (1.82 +/- 0.33, mean +/- SD), and in patients with global myocardial dysfunction (combined data for 20 patients: 1.89 +/- 0.32) than in normal volunteers (2.96 +/- 0.59). These results are similar to previous studies. CONCLUSION: Rapid-sequence 31P-MRS may be a valid diagnostic tool for patients with cardiac disease.

Xanthine oxidase inhibition reduces reactive nitrogen species production in COPD airways.

Reactive nitrogen species (RNS) have been reported to be involved in the inflammatory process in chronic obstructive pulmonary disease (COPD). However, there are no studies on the modulation of RNS in COPD. It was hypothesised that inhibition of xanthine oxidase (XO) might decrease RNS production in COPD airways through the suppression of superoxide anion production. Ten COPD and six healthy subjects participated in the study. The XO inhibitor allopurinol (300 mg x day(-1) p.o. for 4 weeks) was administered to COPD patients. RNS production in the airway was assessed by 3-nitrotyrosine immunoreactivity and enzymic activity of XO in induced sputum as well as by exhaled nitric oxide (eNO) concentration. XO activity in the airway was significantly elevated in COPD compared with healthy subjects. Allopurinol administration to COPD subjects significantly decreased XO activity and nitrotyrosine formation. In contrast, eNO concentration was significantly increased by allopurinol administration. These results suggest that oral administration of the xanthine oxidase inhibitor allopurinol reduces airway reactive nitrogen species production in chronic obstructive pulmonary disease subjects. This intervention may be useful in the future management of chronic obstructive pulmonary disease.

Involvement of the JNK-like protein of the Aedes albopictus mosquito cell line, C6/36, in phagocytosis, endocytosis and infection of West Nile virus.

We recently cloned a c-Jun amino-terminal kinase (JNK) sequence from the C6/36 cell line, derived from the mosquito Aedes albopictus. We showed that SP600125, an inhibitor of JNK proteins, inhibits phagocytosis by C6/36 cells, suggesting that the JNK-like protein regulates phagocytosis. Here, we show that C6/36 cells constitutively express low levels of mRNA encoding the antibacterial peptides, cecropin and defensin, but that these mRNAs were up-regulated upon stimulation by lipopolysaccharide (LPS). Thus, the C6/36 cells have properties similar to those of mammalian macrophages. To characterize further the functional properties of C6/36 cells, we have assayed the role of the JNK-like protein in phagocytosis, endocytosis, and viral infection. C6/36 cells phagocytosed bacteria and artificial beads, and this was only slightly up-regulated following LPS stimulation, suggesting that newly stimulated JNK-like protein was not necessary for phagocytosis. SP600125 inhibited the acidification of intracellular compartments, including those involved in the endocytic pathway. Pretreatment of C6/36 cells with SP600125 or bafilomycin A1, but not cytochalasin D, inhibited the entry of West Nile virus (WNV), suggesting that WNV is internalized mainly by endocytosis, and that the JNK signalling pathway is important for endocytic entry. These findings indicate that the JNK-like protein regulates basic physiological functions, including phagocytosis and endocytosis and infection of WNV.

Correlation between change in pulmonary function and suppression of reactive nitrogen species production following steroid treatment in COPD.

BACKGROUND: Reactive nitrogen species (RNS) have a number of inflammatory actions and the production of these molecules has been reported to be increased in the airways of patients with chronic obstructive pulmonary disease (COPD), which suggests that they may be involved in the inflammatory and obstructive process in COPD. METHODS: The relationship between the reduction in RNS and the improvement in pulmonary function was studied in 18 patients with COPD following steroid treatment (800 micro g beclomethasone dipropionate inhalation for 4 weeks). Twelve patients were treated with inhaled steroids and the others received placebo treatment. Forced expiratory volume in 1 second (FEV(1)) and airway responsiveness to histamine were measured before and after treatment. Induced sputum cells were stained with anti-nitrotyrosine antibody, a footprint of RNS, and RNS formation was assessed by measuring nitrotyrosine immunoreactivity. The immunoreactivity of inducible nitric oxide synthase (iNOS) in induced sputum and exhaled NO levels were also measured. RESULTS: Treatment with steroids resulted in a significant reduction in both nitrotyrosine and iNOS immunoreactivity in sputum cells compared with pretreatment levels (both p<0.01). The reduction rates in both parameters were significantly related (p<0.05). The reduction in nitrotyrosine and iNOS immunoreactivity was correlated with the improvement in FEV(1) (p<0.05) and airway responsiveness to histamine (p<0.01). None of the parameters was significantly changed by placebo administration. CONCLUSIONS: These results suggest that RNS may be involved in the reversible component of inflammation in COPD that is suppressed by steroids. Further studies using specific inhibitors for RNS are needed to clarify their effects on the long term progression of COPD.

The involvement of matrix metalloproteinases in basement membrane injury in a murine model of acute allergic airway inflammation.

BACKGROUND: Airway remodelling in asthma such as subepithelial fibrosis is thought to be the repair process that follows the continuing injury as of chronic airway inflammation. However, how acute allergic inflammation causes tissue injury in the epithelial basement membrane in asthmatic airways remains unclear. Matrix metalloproteinases (MMPs) capable of degrading almost all of the extracellular matrix components have been demonstrated to be involved in cell migration through the basement membrane in vivo and in vitro. OBJECTIVE: We investigated the alterations of matrix construction and the role of MMPs in matrix degradation in the subepithelium during acute allergic airway inflammation. METHODS: Airway inflammation, the ultrastructure of the subepithelium and injury of types III and IV collagen in tracheal tissues from ovalbumin (OVA)-sensitized mice after OVA inhalation with or without the administration of tissue inhibitor of metalloproteinase-2 (TIMP-2) and dexamethasone were evaluated by cell counting in bronchoalveolar lavage (BAL) fluids, electron microscopy and immunohistochemistry, respectively. RESULTS: The disruption of the lamina densa and matrix construction and the decrease of the immunoreactivity for type IV collagen in subepithelium were observed in association with the accumulation of inflammatory cells in airways 3 days after OVA inhalation. This disorganization of the matrix components in the subepithelium, as well the cellular accumulation, was abolished by the administration of TIMP-2 and dexamethasone. The immunoreactivity for type IV collagen in the subepithelium in OVA-inhaled mice returned to the level of that in saline-inhaled mice 10 days after inhalation in association with a decrease of the cell numbers in the BAL fluid. The immunoreactivity for type III collagen was changed neither 3 nor 10 days after OVA inhalation. CONCLUSION: These results suggest that epithelial basement membrane gets injured by, at least in part, MMPs as a consequence of cell transmigration through the membrane during acute allergic airway inflammation.

iNOS depletion completely diminishes reactive nitrogen-species formation after an allergic response.

Nitric oxide (NO) shows proinflammatory actions mainly via reactive nitrogen species (RNS) formation through superoxide- and peroxidase-dependent mechanisms. The purpose of this study was to examine the role of inducible NO synthase (iNOS) in RNS production, airway hyperresponsiveness, and inflammation after allergen challenge. Ovalbumin (OVA)-sensitised, iNOS-deficient and wild-type mice were used. RNS production was assessed by nitrotyrosine (NT) immunoreactivity in the airways. Airway inflammation and responsiveness were evaluated by eosinophil accumulation and methacholine (i.v.) challenge, respectively. In wild-type mice, OVA-inhalation challenge increased iNOS immunoreactivity in airway epithelial cells as well as iNOS protein measured by Western blotting. The total amounts of nitrite and nitrate in bronchoalveolar lavage (BAL) fluid were increased, and NT immunoreactivity was also observed abundantly in airway inflammatory cells. In iNOS-deficient mice, both iNOS expression and NT formation were completely abolished, and the total amounts of nitrite and nitrate in BAL fluid were significantly decreased. In contrast, OVA-induced airway eosinophil recruitment and hyperresponsiveness were observed almost equally in wild-type and iNOS-deficient mice. These data suggest that reactive nitrogen species production after allergic reaction occurs totally via inducible nitric oxide synthase-dependent pathways. Allergen-mediated airway eosinophil recruitment and hyperresponsiveness appear to be independent of reactive nitrogen species production.

Proliferative effects of eosinophil lysates on cultured human airway smooth muscle cells.

BACKGROUND: The hypertrophy/hyperplasia of airway smooth muscle (ASM) cells is one of the characteristic features of bronchial asthma. This structural change leads to the thickening of airway walls resulting in the amplification of airway narrowing. However, the pathogenesis of this structural change has not yet been determined. Eosinophils, which play a pathogenic role in asthma, have been demonstrated to have proliferative effects on fibroblasts and vascular smooth muscle cells. OBJECTIVE: We attempted to investigate the potential of eosinophils to induce the proliferation of ASM cells. METHODS: We examined the effect of lysates of eosinophils purified from peripheral blood of healthy donors on cultured human ASM cell proliferation. RESULTS: Eosinophil lysates significantly induced ASM cell proliferation in time- and dose-dependent manners, reaching a maximum on day 6 at 50% of eosinophil lysates (6.0 +/- 0.7 x 104 [mean +/- SD] /well, n = 5 vs. 4.5 +/- 1.1 x 104/well, n = 5; P < 0.05). This proliferative activity was heat-sensitive and recovered in the soluble fraction of the eosinophil lysates. Furthermore, the molecular weight of the mitogenic activity in the soluble fraction was identified as lower than 10 kDa. The inhibitory activity to ASM cell proliferation was also found in the insoluble fraction of the lysates. CONCLUSION: These results indicate that circulating eosinophils store mitogenic activity for ASM cells, suggesting that eosinophils might contribute to the development of the hyperplasia of ASM cells in asthmatics through the release of the stored mitogenic activity upon stimulation at the site of inflammation.

Hypoxia induces transcription factor ETS-1 via the activity of hypoxia-inducible factor-1.

ETS-1 plays an important role in angiogenesis and cancer invasion, and hypoxia is a common feature in these phenomena. We examined whether hypoxia influenced ETS-1 expression. Hypoxia induced ETS-1 in a human bladder cancer cell line, T24, and promoter analysis revealed that the deletion of -424 to -279 bp from the human ETS-1 promoter decreased the hypoxia-mediated inducibility. This region contained a hypoxia responsive element-like sequence, and HIF-1 bound to it under the hypoxic condition. Double-stranded synthetic oligonucleotides of this sequence as a decoy inhibited the hypoxia-mediated inducibility. These results indicate that hypoxia induces ETS-1 via the activity of HIF-1.

Declining trend in the in-hospital case-fatality rate from acute myocardial infarction in Miyagi Prefecture from 1980 to 1999.

The case-fatality rate from acute myocardial infarction (AMI) appears to have been declining in recent decades, so the present study reviewed the trend in in-hospital case-fatalities from AMI in Miyagi Prefecture, Japan, 1980-1999. The causes of death and the effects of gender and age on the trend were also analyzed. From the AMI registration database of the Miyagi Study Group for AMI, 12,961 cases of AMI were analyzed. The 30-day in-hospital case-fatality was calculated from the data for 1980-1999: data for causes of death were available for 1980-1997, and the data concerning primary percutaneous transluminal coronary angioplasty (PTCA) for AMI were available for 1997-1999. The in-hospital case-fatality rate declined from 17.0% in the early 80s to 7.3% in the late 90s (approximately 57% reduction). The in-hospital case-fatality rate was higher in female patients. Rhythm failure substantially decreased in the late 1980s. Pump failure is decreasing, but is still the biggest problem. The in-hospital case-fatality rate was significantly lower in patients received PTCA. The declining trend in the in-hospital case-fatality rate suggests the benefits of current therapeutic procedures, including primary PTCA, for AMI. Pump failure is an important target for further decreasing the trend.

[18F] labeled diacylglycerol analogue as a potential agent to trace myocardial phosphoinositide metabolism.

Phosphoinositide metabolism plays an important role in cardiac pathophysiology. To investigate whether [18F]diacylglycerol could be used to trace myocardial phosphoinositide metabolism, lipids were extracted from rat myocardium after the injection. 1-[8-[18F]fluorooctanoyl]-2-palmitoylglycerol and 1-[8-[18F]fluoropalmitoyl]-2-palmitoylglycerol were predominantly metabolized to phosphatidylethanolamine and triacylglycerol, respectively. The radioactivity incorporated into phosphoinositide metabolism was 51, 44, 32, and 30% 3, 5, 10, and 30 minutes after the injection of 1-[4-[18F]fluorobutyryl]-2-palmitoylglycerol, respectively. 1-[4-[18F]fluorobutyryl]-2-palmitoylglycerol might be a potential tracer to evaluate myocardial phosphoinositide metabolism early after the injection.

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries.

The contribution of the Na+/Ca2+ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (ASK) with pronounced myogenic tone and mesenteric small arteries (AMS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na+/Ca2+ exchanger, the extracellular Na+ concentration ([Na+]o) was lowered from 143 to 1.2 mM by substituting choline-Cl for NaCl. The ASK developed significant myogenic tone and constricted further in low [Na+]o. Nifedipine (1 microM) reduced both myogenic tone and low [Na+]o-induced contraction. Because the membrane potential of ASK was not changed by low [Na+]o (-35 +/- 2 mV at 143 mM [Na+]o, -37 +/- 3 mV at 1.2 mM [Na+]o), depolarization-induced Ca2+ influx was not a cause of the low [Na+]o-induced contraction. The AMS did not develop significant myogenic tone. Although low [Na+]o also constricted AMS, the magnitude of constriction was significantly weaker than that in ASK (17 +/- 4 vs. 47 +/- 6%, P < 0.01, at 58 mM Na+). With Bay K 8644, AMS developed myogenic tone, and low [Na+]o-induced constriction was significantly increased. In conclusion, Na+/Ca2+ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca2+-extrusion.

Role of GABA within the nucleus tractus solitarii in the hypoxic ventilatory decline of awake rats.

The purpose of this study was to examine our hypothesis that gamma-aminobutyric acid (GABA) in the nucleus tractus solitarii (NTS) may be related to the hypoxic ventilatory decline (HVD) and that chemoreceptor stimulation was essential to activate this mechanism. We used unanesthetized, freely moving rats in this study. An in vivo microdialysis technique was used to measure the extracellular GABA concentration ([GABA]o), and an in vivo microinjection technique was used to examine the effects of the GABA agonists and antagonists on the ventilation during hypoxia. The GABA agonists injected into the NTS attenuated the ventilation during hypoxia. By hypoxic exposure, [GABA]o was increased during the HVD. However, by carotid body denervation (CBD), this GABA increase was abolished. Although GABA antagonists microinjected into the NTS during the HVD phase significantly increased the depressed ventilation, this effect on the ventilation was abolished by CBD. These results suggest that the GABA in the NTS has a pivotal role in the HVD and that this mechanism is not activated without chemoreceptor stimulation.

Stretch and quick release of rat cardiac trabeculae accelerates Ca2+ waves and triggered propagated contractions.

Rapid shortening of active cardiac muscle [quick release (QR)] dissociates Ca2+ from myofilaments. We studied, using muscle stretches and QR, whether Ca2+ dissociation affects triggered propagated contractions (TPCs) and Ca2+ waves. The intracellular Ca2+ concentration was measured by a SIT camera in right ventricular trabeculae dissected from rat hearts loaded with fura 2 salt, force was measured by a silicon strain gauge, and sarcomere length was measured by laser diffraction while a servomotor controlled muscle length. TPCs (n = 27) were induced at 28 degrees C by stimulus trains (7.5 s at 2.65 +/- 0.13 Hz) at an extracellular Ca2+ concentration ([Ca2+]o) = 2.0 mM or with 10 microM Gd3+ at [Ca2+]o = 5.2 +/- 0.73 mM. QR during twitch relaxation after a 10% stretch for 100-200 ms reduced both the time between the last stimulus and the peak TPC (PeakTPC) and the time between the last stimulus and peak Ca2+ wave (PeakCW) and increased PeakTPC and PeakCW (n = 13) as well as the propagation velocity (Vprop; n = 8). Active force during stretch also increased Vprop (r = 0.84, n = 12, P < 0.01), but Gd3+ had no effect (n = 5). These results suggest that Ca2+ dissociation by QR during relaxation accelerates the initiation and propagation of Ca2+ waves.

Increased expression of heme oxygenase-1 and bilirubin accumulation in foam cells of rabbit atherosclerotic lesions.

Heme oxygenase-1 (HO-1) catalyzes the regiospecific oxidative degradation of heme to biliverdin IXalpha, iron, and carbon monoxide. Biliverdin IXalpha is subsequently reduced to bilirubin IXalpha by biliverdin reductase. HO-1 expression is induced under various disease conditions, including atherosclerosis, but it is unknown whether HO-1 catalyzes heme breakdown in the regions at risk. Using hypercholesterolemic rabbits fed a cholesterol-enriched diet, we attempted to demonstrate the involvement of HO-1 induction and bilirubin IXalpha production in atherosclerotic regions. Expression levels of HO-1 mRNA were elevated in the aortas of hypercholesterolemic rabbits. In situ hybridization and immunohistochemistry revealed that mRNA and protein of HO-1 are induced in endothelial cells and foam cells (lipid-filled macrophages) in atherosclerotic lesions. Furthermore, immunohistochemistry with the use of an anti-bilirubin-IXalpha monoclonal antibody, 24G7, demonstrated accumulation of bilirubin IXalpha in foam cells, indicating that heme is actually degraded in atherosclerotic lesions. Remarkably, bilirubin IXalpha, like HO-1 protein, is predominantly accumulated in the perinuclear regions of foam cells. These results provide the first in vivo evidence of the colocalization of HO-1 and bilirubin IXalpha in foam cells, suggesting a role of HO-1 induction in the modulation of macrophage activation in atherosclerosis.

Interaction between sarcomere and mitochondrial length in normoxic and hypoxic rat ventricular papillary muscles.

We hypothesized that the mitochondrial length may be altered according to changes in the sarcomere length, and that this relationship may be affected by exposure to hypoxia. Rat ventricular papillary muscles were isolated and immersed in normoxic or hypoxic solutions for 10 min. Sarcomeres of various lengths were obtained by fixing the papillary muscles in a slack or stretched state, or after exposure to a contracture solution containing saponin and CaCl(2). The mitochondrial length measured using electron microscopy significantly correlated to the length of the adjacent sarcomere in both the normoxic (n=767) and hypoxic (n=1145) groups (P<.0001). The slope of the regression line, however, was significantly less steep, and its intercept was significantly larger in the hypoxic group than in the normoxic group (analysis of covariance). When we analyzed the mitochondrial lengths among the three sarcomere-length subgroups (<1.5, 1.5-2.0, and >2.0 microm), the mitochondrial length was significantly shorter in the hypoxic condition than in the normoxic condition at sarcomere lengths greater than 2.0 microm. Staining for desmin, the major muscle-type intermediate filament, the longitudinal system of which connects the mitochondria with the Z bands of sarcomeres, showed a clear cross-striation pattern in both papillary muscles with and without the exposure to hypoxia, suggesting that desmin was preserved after the exposure to hypoxia. These data indicate that the mitochondrial length changes according to changes in the sarcomere length, suggesting the possible role of mitochondria as an internal load against myocyte contraction. It is also suggested that mitochondria exposed to hypoxia may be more resistive to both compression and stretch in a longitudinal direction than those in the normoxic condition.

Gene expression and in situ localization of diacylglycerol kinase isozymes in normal and infarcted rat hearts: effects of captopril treatment.

Diacylglycerol (DG) kinase (DGK) terminates signaling from DG, which serves as an activator of protein kinase C (PKC), by converting DG to phosphatidic acid. DGK is thus regarded as an attenuator of the PKC activity. In rats, five DGK isozymes have been cloned, but little is known about their role in the heart. In this study, the spatiotemporal expression of DGK isozymes was investigated in rat hearts under a normal condition and after myocardial infarction (MI) by in situ hybridization histochemistry and immunohistochemistry. In normal left ventricular myocardium, DGKalpha, DGKepsilon, and DGKzeta mRNAs were expressed evenly throughout the myocardium, although the DGKalpha expression was very low. In infarcted hearts, the expression of DGKzeta was enhanced in the peripheral zone of the necrotic area and at the border zone 3 and 7 days after MI, and to a lesser extent in the middle layer of the granulation tissue 21 days after MI. The enhanced DGKzeta expression in the infarcted and border areas could be attributed to granulocytes and macrophages. In contrast, the expression of DGKepsilon in the infarcted and border areas was lower than that in the viable left ventricle (LV) throughout the postoperation period. Furthermore, DGKepsilon expression in the viable myocardium 21 days after MI decreased significantly compared with left ventricular myocardium in the sham-operated rats and was completely restored by treatment with captopril. Our results demonstrate that three DGK isozymes are expressed in the heart and that each isozyme might have different functional characteristics in the healing and LV remodeling after MI.

Cultured rat glomerular epithelial cells show gene expression and production of transforming growth factor-beta: expression is enhanced by thrombin.

BACKGROUND: Glomerular crescents play an important role in progressive glomerular injury. The lesions consist of epithelial cells, macrophages, and deposits of fibrin and extracellular matrix. Transforming growth factor beta (TGF-beta) contributes to the modulation of cell growth and extracellular matrix synthesis. Thrombin is involved in fibrin formation in crescents. The purpose of this study was to examine whether glomerular epithelial cells (GEC) could produce TGF-beta, and if so, to clarify the role of TGF-beta in GEC proliferation. We also investigated whether thrombin could modulate the production of TGF-beta and extracellular matrix by GEC. METHODS: Bioassay using the TGF-beta-dependent mink pulmonary epithelial cell line (CCL-64), immunoblot analysis, and reverse transcriptase polymerase chain reaction (RT-PCR) were used to demonstrate TGF-beta production by rat GEC. TGF-beta gene expression was examined by RT-PCR in GEC incubated with thrombin, and type IV collagen and fibronectin were quantified by enzyme immunoassay in culture supernatants of GEC incubated with thrombin or TGF-beta. RESULTS: TGF-beta activity was demonstrated in GEC supernatants by bioassay. Immunoblot analysis of concentrated culture supernatants using anti-TGF-beta antibody revealed a 12.5-kDa protein, which was compatible with TGF-beta. Concentrated GEC supernatants inhibited GEC proliferation as well as porcine TGF-beta. RT-PCR demonstrated TGF-beta gene expression in GEC. Thrombin (0.5-5.0 U/ml) enhanced TGF-beta mRNA expression in a dose-dependent manner. Thrombin (5.0 U/ml) and porcine TGF-beta (5.0 ng/ml) stimulated the production of type IV collagen and fibronectin by GEC. CONCLUSIONS: Rat GEC produce TGF-beta in vitro. Thrombin may participate in the progression of glomerulosclerosis in crescentic glomerulonephritis through the stimulation of TGF-beta production by GEC.

Nonselective endothelin receptor antagonist initiated soon after the onset of myocardial infarction may deteriorate 24-hour survival.

To investigate the effects of endothelin blockade initiated immediately after the onset of myocardial infarction on survival and left ventricular remodeling, treatment with the nonselective receptor antagonist TAK-044 (n = 22) or saline (n = 19) for 3 weeks was initiated immediately after coronary ligation in rats. The 24-h survival rate was significantly lower in the TAK-044 group than in the saline group. The systolic blood pressure 24 h after the onset of myocardial infarction was similar in the saline and TAK-044 groups, although it was significantly lower in the TAK-044 group during the 3-week protocol. Heart weight/tibial length was significantly increased in the TAK-044 group compared with the saline group. As all deaths in the TAK-044 group occurred within 24 h after myocardial infarction, we performed additional experiments using a separate group of rats 12-16 h after myocardial infarction. Plasma and myocardial endothelin-1 levels were significantly increased, and a bolus injection of TAK-044 significantly reduced left ventricular dP/dtmax in these rats that had had a myocardial infarction compared with sham-operated rats. Endothelin receptor blockade initiated immediately after the onset of myocardial infarction may deteriorate acute-phase survival and left ventricular remodeling. Inhibition of the positive inotropic action of endothlin-1 may partially explain the increased 24-h mortality.

Microvolt T wave alternans in human cardiac hypertrophy: electrical instability and abnormal myocardial arrangement.

INTRODUCTION: Although T wave alternans (TWA) is a promising risk marker for myocardial electrical instability, it remains unclear how the presence of TWA is related to myocardial damage. METHODS AND RESULTS: TWA was measured in 28 patients with hypertrophic cardiomyopathy (HCM), 29 patients with hypertensive left ventricular hypertrophy (HLVH), and 15 normal volunteers using a CH2000 system. The amplitude of TWA (Valt) was measured at the lead with the maximum amplitude. Cardiac biopsy was performed in 12 HCM patients, who were divided into two groups (severe and mild) based on histologic findings of myocardial disarray and fibrosis. TWA was positive (Valt > 1.9 microV) in 61% of HCM and 31% of HLVH, despite a nearly identical left ventricular mass index (176 +/- 65 g/m2 vs 175 +/- 39 g/m2). Valt at heart rate = 110 beats/min was significantly greater in HCM with severe disarray and fibrosis than in HCM with mild disarray and in HLVH. CONCLUSION: In HCM patients, a positive TWA test probably is related to abnormal myocardial arrangement (disarray) and/or fibrosis, and it may reflect electrical instability of the myocardium.