内皮素-1对培养新生大鼠心肌细胞原癌基因c-fos表达的诱导

本实验用无血清的培养新生大鼠心肌细胞,探讨内皮素１（ＥＴ１）对原癌基因ｃｆｏｓ表达的作用。结果显示：ＥＴ１可显著诱导ｃｆｏｓ的表达,其表达的高峰在３０ｍｉｎ,２ｈ恢复到正常水平,并呈剂量依赖性反应和被ＥＴＡ的特异性受体拮抗剂ＢＱ１２３所阻断;蛋白激酶Ｃ（ＰＫＣ）激动剂ＰＭＡ可诱导ｃｆｏｓ表达,而ＰＫＣ抑制剂Ｓｔａｕｒｏｓｐｏｒｉｎｅ则可阻断ＥＴ１诱导的ｃｆｏｓ表达;钙通道阻断剂硝苯吡啶预处理心肌细胞对ＥＴ１诱导的心肌细胞的ｃｆｏｓ表达无明显的作用。这些结果提示,ＥＴ１诱导ｃｆｏｓ表达是通过ＥＴＡ受体介导的,ＰＫＣ在此过程中起重要作用。     

Heterogeneity in MT1-MMP activity with ischemia-reperfusion and previous myocardial infarction: relation to regional myocardial function

After a myocardial infarction (MI), an episode of ischemia-reperfusion (I/R) can result in a greater impairment of left ventricular (LV) regional function (LVRF) than that caused by an initial I/R episode in the absence of MI. Membrane type-I matrix metalloproteinase (MT1-MMP) proteolytically processes the myocardial matrix and is upregulated in LV failure. This study tested the central hypothesis that a differential induction of MT1-MMP occurs and is related to LVRF after I/R in the context of a previous MI. Pigs with a previous MI [3 wk postligation of the left circumflex artery (LCx)] or no MI were randomized to undergo I/R [60-min/120-min left anterior descending coronary artery (LAD) occlusion] or no I/R as follows: no MI and no I/R (n = 6), no MI and I/R (n = 8), MI and no I/R (n = 8), and MI and I/R (n = 8). Baseline LVRF (regional stroke work, sonomicrometry) was lower in the LAD region in the MI group compared with no MI (103 ± 12 vs. 188 ± 26 mmHg·mm, P < 0.05) and remained lower with peak ischemia (35 ± 8 vs. 88 ± 17 mmHg·mm, P < 0.05). Using a novel interstitial microdialysis method, MT1-MMP was directly measured and was over threefold higher in the LCx region and over twofold higher in the LAD region in the MI group compared with the no MI group at baseline. MT1-MMP fluorogenic activity was persistently elevated in the LCx region in the MI and I/R group but remained unchanged in the LAD region. In contrast, no changes in MT1-MMP occurred in the LCx region in the no MI and I/R group but increased in the LAD region. MT1-MMP mRNA was increased by over threefold in the MI region in the MI and I/R group. In conclusion, these findings demonstrate that a heterogeneous response in MT1-MMP activity likely contributes to regional dysfunction with I/R and that a subsequent episode of I/R activates a proteolytic cascade within the MI region that may contribute to a continued adverse remodeling process.     

Mechanics of the left ventricular myocardial interstitium: effects of acute and chronic myocardial edema

Myocardial interstitial edema forms as a result of several disease states and clinical interventions. Acute myocardial interstitial edema is associated with compromised systolic and diastolic cardiac function and increased stiffness of the left ventricular chamber. Formation of chronic myocardial interstitial edema results in deposition of interstitial collagen, which causes interstitial fibrosis. To assess the effect of myocardial interstitial edema on the mechanical properties of the left ventricle and the myocardial interstitium, we induced acute and chronic interstitial edema in dogs. Acute myocardial edema was generated by coronary sinus pressure elevation, while chronic myocardial edema was generated by chronic pulmonary artery banding. The pressure-volume relationships of the left ventricular myocardial interstitium and left ventricular chamber for control animals were compared with acutely and chronically edematous animals. Collagen content of nonedematous and chronically edematous animals was also compared. Generating acute myocardial interstitial edema resulted in decreased left ventricular chamber compliance compared with nonedematous animals. With chronic edema, the primary form of collagen changed from type I to III. Left ventricular chamber compliance in animals made chronically edematous was significantly higher than nonedematous animals. The change in primary collagen type secondary to chronic left ventricular myocardial interstitial edema provides direct evidence for structural remodeling. The resulting functional adaptation allows the chronically edematous heart to maintain left ventricular chamber compliance when challenged with acute edema, thus preserving cardiac function over a wide range of interstitial fluid pressures.     

Expression of myoblast and myocyte antigens in relation to differentiation and the cell cycle

Cell cycle parameters and expression of myoblast and myocyte antigens were investigated during exponential growth and during the differentiation phase of rat L8( E63 ) myoblasts by an integrated approach involving microspectrophotometry with DNA fluorochromes, [3H]thymidine autoradiography, and immunofluorescent staining with monoclonal antibodies. In addition to the majority of cells which are recruited into myotubes, two distinct populations of mononucleate cells were resolved in cultures of rat myoblasts undergoing differentiation. These mononucleate cells consist of (1) a population of proliferating cells with a prolonged G1 transit time; (2) a population of non-proliferating cells which remain arrested in G1 for more than 72 h. The latter group was examined with respect to the expression of two marker antigens recognized by two monoclonal antibodies: antibody B58 reacts with a macromolecular component present in undifferentiated myoblasts but not in mature myotubes, and antibody XMlb reacts with a muscle-specific isoform of myosin. All four possible combinations of expression of these antigens by single cells were found: B58 +XM1b -, B58 +XM1b +, B58 - XM1b -, and B58 - XMlb +. The implication of these findings with respect to the transition from the proliferative to the differentiative phase of myogenesis is discussed.     

Vascular remodeling and ET-1 expression in rat strains with different responses to chronic hypoxia

Chronic hypoxia leads to a greater degree of pulmonary hypertension in the Wistar-Kyoto (WKY) rat than in the Fischer 344 (F-344) rat. We questioned whether this difference is associated with baseline differences in pulmonary artery anatomy, a greater degree of hypoxia-induced pulmonary vascular remodeling in the WKY rat, and/or differences in expression of endothelin (ET)-1. Male F-344 and WKY rats were maintained in normoxia or normobaric hypoxia for 21 days. Morphometry revealed that baseline pulmonary artery anatomy was similar in the two strains. However, during chronic hypoxia, the WKY rats developed a greater degree of muscularization of small pulmonary arteries. Baseline plasma and lung immunoreactive ET-1 levels were similar in the WKY and F-344 rats and increased significantly during hypoxia in the WKY rats. Northern analysis demonstrated increased lung preproET-1 mRNA during hypoxia in both strains, with a greater increase in WKY rats. Immunostaining demonstrated increased ET-1 in bronchial epithelium and peripheral pulmonary arteries during hypoxia, although to a greater degree in the WKY rats. We conclude that the WKY strain demonstrates increased susceptibility to hypoxia-induced pulmonary vascular remodeling compared with the F-344 strain and that increased lung and circulating ET-1 levels during hypoxia may partly explain this difference.     

Rac1 activity is required for cardiac myocyte alignment in response to mechanical stress

Mechanical stretch is essential for the cardiac growth. The exposure of cardiac myocytes to the mechanical stretch leads to the cell alignment in parallel to the stretch direction, determining the cell polarity, though it remains to be addressed how mechanical stretch regulates cell orientation. In the present study, we investigated the signal transduction pathways responsible for the cell orientation response to mechanical stretch, focusing on Rho family proteins. Neonatal rat cardiomyocytes were cultured on silicon chambers and exposed to artificial uniaxial cyclic stretch. The pull-down assays revealed that Rac1 was rapidly activated by stretch, but not RhoA. To analyze the roles of Rho family proteins in cardiomyocyte orientation, adenoviral vectors expressing dominant-negative (dn) RhoA and Rac1 were generated. The transfection with adenovirus vector expressing dnRac1, but not dnRhoA, inhibited stretch-induced cell alignment. In conclusion, Rac1 activity is necessary for cardiomyocyte alignment in response to directional stretch.     

Insulin sensitivity and big ET-1 conversion to ET-1 after ETA- or ETB-receptor blockade in humans.

Cardiovascular diseases are characterized by insulin resistance and elevated endothelin (ET)-1 levels. Furthermore, ET-1 induces insulin resistance. To elucidate this mechanism, six healthy subjects were studied during a hyperinsulinemic euglycemic clamp during infusion of (the ET-1 precursor) big ET-1 alone or after ET(A)- or ET(B)-receptor blockade. Insulin levels rose after big ET-1 with or without the ET(B) antagonist BQ-788 (P < 0.05) but were unchanged after the ET(A) antagonist BQ-123 + big ET-1. Infused glucose divided by insulin fell after big ET-1 with or without BQ-788 (P < 0.05). Insulin and infused glucose divided by insulin values were normalized by ET(A) blockade. Mean arterial blood pressure rose during big ET-1 with or without BQ-788 (P < 0.001) but was unchanged after BQ-123. Skeletal muscle, splanchnic, and renal blood flow responses to big ET-1 were abolished by BQ-123. ET-1 levels rose after big ET-1 (P < 0.01) in a similar way after BQ-123 or BQ-788, despite higher elimination capacity after ET(A) blockade. In conclusion, ET-1-induced reduction in insulin sensitivity and clearance as well as splanchnic and renal vasoconstriction are ET(A) mediated. ET(A)-receptor stimulation seems to inhibit the conversion of big ET-1 to ET-1.     

内皮素对培养心肌细胞内游离钙浓度的作用

实验用培养新生ＳＤ大鼠心室肌细胞,以Ｆｕｒａ－２／ＡＭ荧光指示剂负载检测收肌细胞内游离钙浓度（「Ｃａ＾２＋」）的变化,探讨内皮素－１（ＥＴ－１）对「Ｃａ＾２＋」ｉ的作用及其机制。结果显示：ＥＴ－１引起心肌细胞「Ｃａ＾２＋」ｉ升高有两个时相,瞬时相持续相。ＥＴ－１诱导的瞬时相「Ｃａ＾２＋」ｉ升高呈浓度依赖性,预先用ＥＴＡ特异性受阻断剂ＢＱ１２３处理,可阻断ＥＴ－１引起的「Ｃａ＾２＋」ｉ升高,揭示上述     

Thrombin activity throughout the acute phase of acute ST-elevation myocardial infarction and the relation to outcome

Background: Thrombin and plasmin play a central role in ongoing thrombosis and platelet activation in patients with acute ST-elevation myocardial infarction (STEMI). Data of thrombin and plasmin activity in the early course of STEMI and the relation to outcome are scarce.

Methods: We included 68 consecutive patients (53 male, 59?±?11.4 years) with STEMI who underwent acute catheter-based reperfusion therapy within the first 12?h after onset of symptoms. Blood samples were taken at admission and after 4, 8, 12 and 24?h. Thrombin activity and generation was measured by changes in the thrombin/antithrombin-III complex (TAT) and prothrombin fragment (F1.2); plasmin was measured by changes in the plasmin-α₂/antiplasmin complex (PAP). A follow-up with respect to the combined primary endpoint consisting of death, acute myocardial infarction or urgent need for revascularization up to 6 weeks post-discharge was carried out.

Results: TAT values showed no significant change over time in patients with and without the primary endpoint but there was a borderline difference between these groups at 4?h after admission (event group 9.0 vs no event group 4.7?μg l^?1, p?=?0.057). F1.2 values were different between groups only after 24?h (event group 1.5 vs no event group 0.9?nmol l^?1, p?=?0.028) and did not differ in serial sampling of 24?h. PAP values were higher in patients with events after 4 and 8?h and declined over time in the group without events (p?<0.001). Odds ratios (OR) with respect to the primary endpoint were highest for TAT?>4.8?μg l^?1 at 0?h and TAT?>8.4?μg?l^?1 at 4?h (OR 7.1, 95% confidence interval (CI) 1.5–34, p?=?0.015 and OR 5.5, 95% CI 1.5–20.0, p?=?0.01, respectively). The predictive value of plasmin concentrations were equally high after 4?h (PAP?>962?μg l^?1; OR 6.8, 95% CI 1.8–26.2, p?=?0.005) and 8?h (PAP?>495?μg l^?1, OR 6.7, 95% CI 1.4–32.9, p?=?0.024). Values for F1.2 were only predictive after 24?h (F1.2?>0.85?nmol l^?1, OR 13, 95% CI 1.4–117.8, p?=?0.023).

Conclusions: Markers of thrombin and plasmin activity in acute STEMI are related to outcome. The marker for thrombin generation F1.2 becomes a significant predictor of outcome at 24?h after admission, reflecting the potentially adverse effects of ongoing thrombin generation. This underlines the potential for direct thrombin inhibition and individualization of treatment by thrombin markers in STEMI.     

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector.     

Overexpression of sarcolemmal calcium pump attenuates induction of cardiac gene expression in response to ET-1

The function of the plasma membrane calmodulin-dependent calcium ATPase (PMCA) in myocardium is unknown. PMCA is localized in caveolae, 50- to 100-nm membrane invaginations, which also contain receptors for endothelin-1 (ET-1) and various other ligands. PMCA has been suggested to play a role in regulation of caveolar signal transduction. We studied the effects of the hypertrophic agonist ET-1 and increased coronary perfusion pressure on cardiac synthesis of B-type natriuretic peptide (BNP) in transgenic rats overexpressing the human PMCA 4CI in isolated perfused heart preparation. ET-1 infusion for 2 h increased BNP mRNA levels twofold in left ventricles (LV) of nontransgenic rats, whereas no increase was noted in PMCA rat hearts. Similar responses were seen in adrenomedullin and c-fos mRNA levels, and in immunoreactive BNP secretion. Increased mechanical load produced by elevated perfusion pressure induced similar 1.5- to 1.6-fold increases in LV BNP mRNA in both nontransgenic and PMCA rat hearts. These results show that cardiac overexpression of PMCA attenuates ET-1-stimulated early induction of cardiac gene expression, suggesting that PMCA may modulate myocardial growth responses.     

c-myc Gene expression is localized to the myocyte following hemodynamic overload in vivo

Expression of the proto-oncogene c-myc increases in the hemodynamically overloaded heart, but expression by cardiac myocytes has not been shown. To address this issue, right ventricular overload was induced in cats by pulmonary artery banding. Expression of c-myc and α-skeletal actin mRNA were determined by Northern analysis. Immuno-reactive Myc protein was identified by histochemical staining. Steady state levels of c-myc mRNA peaked within 2 h after banding. Levels of α-skeletal actin mRNA were maximally increased 48 h–1 week after banding and were still elevated at 1 month. Prominent staining of myocyte nuclei for immunoreactive Myc protein was detected 48 h after banding although a few interstitial nuclei were also positive. These studies show that c-myc and α-skeletal actin gene expression are upregulated in a large animal model of hemodynamic overload. The localization of the immunoreactive Myc protein to right ventricular myocyte nuclei after pulmonary artery banding supports the hypothesis that c-myc induction is part of a general response in cardiac hypertrophy that is common to many mammalian species.     

Increased activity and expression of histone deacetylase 1 in relation to tumor necrosis factor-alpha in synovial tissue of rheumatoid arthritis

Introduction  

The purpose of this study was to investigate the profile of histone deacetylase (HDAC) expression in the synovial tissue of rheumatoid arthritis (RA) compared with that of normal control and osteoarthritis (OA), and to examine whether there is a link between HDAC activity and synovial inflammation.     

Reaction patterns of the myocardial interstitium in different models of cardiac hypertrophy--stereological investigations

Several experimental models of cardiac hypertrophy were investigated in rats: 1. mild hypertrophy induced by physical exercise (18 weeks), 2. mild hypertrophy induced by renovascular hypertension (24 weeks), 3. moderate hypertrophy induced by renovascular hypertension in diabetic and non-diabetic animals (8 weeks), 4. moderate hypertrophy induced by renovascular hypertension in diabetic and non-diabetic animals (12 weeks), 5. moderate hypertrophy induced by thyroxin application (4 weeks), 6. mild hypertrophy in chronic uremia (5/6 nephrectomy, 3 weeks). It is concluded from quantitative stereological parameters of the left ventricular papillary muscles that 1. in hypertrophic hearts myocardial blood flow and oxygen consumption, respectively, rather than the size of muscle fibres determine the capillary supply of the myocardium, 2. interstitial fibrosis occurs in hypertrophy induced by chronic pressure overload and depends on degree and duration of hypertension, 3. the extent of interstitial fibrosis in hypertension is magnified by diabetes mellitus, and 4. the interstitial fibrosis which occurs in chronic uremia is not caused by hypertension.     

Zinc-metalloproteases in insects: ACE and ECE

Research on the angiotensin-converting enzyme (ACE) in insects has substantially advanced during the recent decade. The cloning of this enzyme in many insect species, the determination of the 3D-structure and several molecular and physiological studies have contributed to the characterization of insect ACE as we know it today: a functional enzyme with a putative role in reproduction, development and defense. The discovery of the endothelin-converting enzyme in insects occurred more recently and cloning of the corresponding cDNA has been carried out in only one insect species so far. However, activity studies and analysis of insect genomes indicate that this enzyme is also widely distributed among insect species. Making hypotheses about its putative function would be preliminary, but its wide tissue distribution suggests a major and diverse biological role.