Ultrastructural stereological analysis of the absolute parameters of the cardiomyocytes in myocardial hypertrophy

Arterial hypertension was produced in male Wistar rats by abdominal aorta ligation. By the 35th day of experiment the animals had developed myocardial hypertrophy. The subcellular organization of cardiomyocytes was studied by electron microscopy and stereology (computation of relative and absolute parameters). In myocardial hypertrophy, the absolute volume of myofibrils, sarcoplasmic reticulum and T system in the left ventricle of the heart was increased, whereas the absolute volume of the mitochondria remained unchanged. The general surface area of all organelles was noticeably increased. The myofibrils and sarcoplasmic reticulum experienced greater changes, which may reflect the features of compensatory processes under the experimental conditions described.     

Effect of embryonal organ-specific RNA on myocardial hypertrophy]

The relative weight of the heart and the thickness of muscular fibers in the left ventricular myocardium of rats were evaluated 70-91 and 120-227 days after creating stenosis of the abdominal portion of the aorta. Half of the animals were inoculated with cardiac type-specific embryonal RNA, the rest of the animals being intact. The data were treated by the variational statistical technique, by dispersion and mathematic analyses. Administration of RNA decreased the rate of myocardial hypertrophy development.     

Prostaglandins, cyclic nucleotides and heart adaptation to acute and chronic pressure overload]

The content of prostaglandins (PGs) and cyclic nucleotides was determined in the rat heart at different time following coarctation of the abdominal aorta. The animals were divided into three groups according to the rate and degree of hypertrophy. The rats with the best adaptability (the highest level of hypertrophy) showed the highest myocardial PG content. An increase in myocardial weight in response to the overload correlated with an enhancement in PGE/PGF2 ratio. The relationship between the myocardial levels of PGs and cyclic nucleotides during the period of adaptation was also observed. Prostaglandins are suggested to play an important role in the heart adaptation to pressure overloads.     

大鼠心脏血压超负荷诱导左心室HSP70基因表达

本工作应用热休克蛋白７０（ＨＳＰ７０）核酸分子杂交方法,测定了大鼠腹主动脉缩窄后左心室ＨＳＰ７０ｍＲＮＡ的水平。结果表明：大鼠腹主动脉缩窄后４ｈ,动脉血压已明显升高,并持续在高水平上;大鼠左心室重／体重比在第三天开始增加,然后持续升高,第４周时比对照组增加５９％;左心室ＨＳＰ７０ｍＲＮＡ在腹主动脉缩窄后４ｈ已明显升高,１ｄ,２ｄ,１ｗ均维持在高水平,１ｗ后逐渐消失。实验结果提示：大鼠心肌负荷增加早期,左心室ＨＳＰ７０ｍＲＮＡ表达明显增加。     

大鼠胸主动脉缩窄诱导心肌肥厚模型的建立

目的建立大鼠胸主动脉部分缩窄诱导心肌肥厚动物模型。方法雄性SD大鼠30只,随机分为两组：胸主动脉缩窄组20只和同期假手术组10只。在右无名动脉和左颈总动脉之间将主动脉结扎于8G针头上,随后将针头退出即可。术后10周,采用超声心动图检测心脏、观察心脏的大体剖面以及HE染色、测量心肌肥厚指数评价心肌肥厚的效果。结果术后10周,肉眼观：模型组心脏体积明显大于对照组。M型超声示：模型组较假手术组缩短分数下降,左室内径和室壁厚度明显增加。超声测量结果示：模型组与假手术组比较：室间隔厚度增加明显（2.527±0.269 vs.1.943±0.1）mm,（P〈0.01）;后壁厚度增加明显（2.492±0.242 vs.1.902±0.076）mm,（P〈0.01）;缩短分数略减小（49±7.681 vs.55.7±9.828）（P〉0.05）;左室舒张末期内径、左室收缩末期内径及射血分数均无明显变化。心脏肥厚指数明显增大（3.196±0.11 vs.1.785±0.099）,P〈0.01。结论胸主动脉缩窄可以导致大鼠心肌肥厚,为研究心室肥厚、心肌功能障碍以及心肌重构提供了一个很好的模型。     

Relationship of cardiac hemodynamic and biochemical adaptations to mortality during long-term aortic constriction

To determine the biochemical and hemodynamic responses to aortic ligation, and to assess the survival rate after the induction of hypertension, 90 normotensive rats were subjected to surgical constriction of the abdominal aorta. Mortality, left ventricular hemodynamics, myocardial biochemical assays, and plasma renin assays were determined 1 week, 1 month, 3 months, or 1 year later. Mortality was greatest between 1 week and 3 months after aortic ligation, during which plasma renin activity was significantly elevated. The rate of left ventricular pressure rise, contractile index, and myocardial alpha-adrenoceptor number were increased at 1 month, but were comparatively depressed at 3 months after the operation, suggesting that the heart was in failure at this time. At 1 year after ligation, hemodynamic and biochemical parameters continued toward normalization. Our data suggest that, in this rodent model, cardiac pump failure occurs through a combination of time-dependent, pressure-induced mechanical adaptations and myocardial biochemical changes that involve both the renin-angiotensin and sympathetic nervous systems. The observed relationship between mortality, myocardial hemodynamics, and biochemical parameters may be used for additional basic research investigations concerning the early periods of cardiac failure.     

The Role of Guanine Nucleotide Binding Proteins in Hamsters with Myocardial Hypertrophy

Abstract

To clarify the role of the guanine nucleotide binding proteins (G-proteins) in the pathogenesis of myocardial hypertrophy, we investigated the alterations in myocardial G proteins in 20-week-old F1b hamsters with pressure overload induced for 3 days (3-day AS), 7 days (7-day AS) and 14 days (14-day AS) by the stenosis of the abdominal aorta, and in 4- and 20 week-old BIO 14.6 Syrian hamsters (4-wk and 20-wk BIO) with genetic myocardial hypertrophy. The hearts of 7-day AS, 14-day AS and 20-wk BIO with left ventricular hypertrophy exhibited a decrease in the mRNA levels detected by Northem blot analysis and protein levels of G protein detected by Western blot analysis as compared with sham-operated and age-matched F1b hearts. The function of Gs or Gi showed a concomitant reduction in both models of myocardial hypertrophy. The hearts of 3-day AS and 4-wk BIO without myocardial hypertrophy showed no changes in G proteins as compared with sham-operated and age-matched F1b hearts. These results suggest that a decrease in G proteins is not involved in the pathogenesis of myocardial hypertrophy, but that myocardial hypertrophy reduced the G proteins.     

Role of adrenergic mechanisms in the development of cardiac hypertrophy.

This experiment was designed to study the role of cardiac beta-adrenergic mechanisms in the development of hypertrophy in rats. The suprarenal abdominal aorta was banded, resulting in an increase in cardiac wt-body wt ratio. A group of rats received a sham operation. Half of the banded rats were treated with practolol, 2.0 mg/kg intraperitoneally every 12 hr for the 6 days after banding. The effectiveness of cardiac beta-adrenergic blockade was confirmed by absence of an increase in heart rate following intravenous isoproterenol at various times between practolol injections. Practolol did not affect the gradient in the banded groups. Six animals in each banded group were sacrificed daily for 6 days. The right and left ventricles were dissected separately and weighed. RV-body weight ratios increased similarly in both banded groups. LV-body weight ratio (g/kg) was 2.17 +/- 0.043 in sham rats, and it attained maximal levels of 3.03 +/- 0.10 within 6 days in banded untreated rats and 2.96 +/- 0.14 in banded rats receiving practolol. Therefore, beta-adrenergic mechanisms were not involved in the development of hypertrophy due to increased afterload. Also, these findings are not consistent with the Meerson hypothesis, since hypertrophy occurred despite the reduction in myocardial O2 consumption due to practolol.     

New experimental rabbit model with PTFE grafts interpositioned in infrarenal abdominal aorta--influence of hyperlipidemia in prosthetic grafts

The major obstacle to clinical application of artificial blood vessel grafts with inside diameter of less than 4 mm is neogenic intimal hypertrophy at anastomotic sites. With the aim of preventing this artificial blood vessel graft anatomotic intimal hypertrophy, attempts have been made to improve surgical techniques and develop new materials for sutures and the grafts themselves. In the assessment of the preventive effects of various measures on anastomotic intimal hypertrophy, it is desirable to minimize variation in preoperative arteriosclerotic changes and uniform hemodynamics after vessel replacement surgery among the subjects. The present authors succeeded in creating an infrarenal abdominal aorta replacement model that meets these requirements using rabbits, and conducted experiments using this model to assess the effects of hyperlipidemia on anastomotic intimal hypertrophy. The anastomotic intimal hypertrophy lesion in the present rabbit infrarenal abdominal aorta replacement model is both morphologically and histologically similar to that found in human artificial blood vessel graft anastomotic sites. In addition, this model permits the easy obtain of animals showing the same hemodynamic status after vascular surgery. For these reasons, the present model is expected to serve well as an experimental model of artificial blood vessel graft anastomotic intimal hypertrophy.     

Cardiac hypertrophy in rats after supravalvular aortic constriction. I. Size and number of cardiomyocytes, endothelial and interstitial cells

The ascending aorta of 22 adult male Sprague-Dawley rats was constricted with a silver ring, and 25 animals were subjected to a sham-operation. The hearts, including the main arteries, were fixed by retrograde perfusion 3, 7, 14, 21 and 35 days after the operation. The cross-sectional area of the aorta was reduced by the constriction to an average of 20% of the values found after sham-operation. Twenty-one days after the constriction the weight of the left ventricular myocardium including the septum was increased 1.7-fold compared with controls. No further increase in weight was observed 35 days after the operation. The relative volumes of the tissue components remained largely constant in the subepicardial myocardium. In the subendocardial myocardium, however, the volume fraction of interstitial and, to a lesser extent, of endothelial tissue was significantly increased. Twenty-one days after constriction the estimated total volumes of the different myocardial components per left ventricle were increased 1.7-fold for heart muscle parenchyma, 1.8-fold for endothelial tissue, 2.9-fold for interstitial tissue, and 1.3-fold for capillary lumina compared with controls. At 35 days, only the interstitial tissue showed a further increase to 4.8-fold of control values. The mean cardiomyocyte volume was increased after aortic constriction in proportion to the increase in left ventricular weight, i.e. 1.7-fold over controls at 21 days. After 35 days its value was 29,500 +/- 790 micron 3 in rats subjected to aortic constriction compared with 16,800 +/- 640 micron 3 in controls. At this time the estimated number of cardiomyocytes per left ventricle showed no significant differences between experimental animals (2.9 X 10(7)) and controls (3.1 X 10(7)). Endothelial and interstitial cells were not only increased in average single cell volume (1.3-fold and 2.0-fold, respectively), but also in number per left ventricle (1.4-fold and 2.7-fold, respectively). Two-dimensional parameters indicated that during hypertrophy the capillary supply lagged behind the overall mass increase but achieved control levels on termination of hypertrophic growth at 35 days. These results show that even in pronounced hypertrophy the increase in mass of the myocardial parenchyma in the rat is due exclusively to an enlargement of cardiomyocytes (hypertrophy), whereas in endothelial and interstitial tissues enlargement of cells as well as increase in cell number (hyperplasia) also plays a role.     

Roles of insulin-like growth factor II in cardiomyoblast apoptosis and in hypertensive rat heart with abdominal aorta ligation

Although IGF-II activating the IGF-II receptor signaling pathway has been found to stimulate cardiomyocyte hypertrophy, the role of IGF-II in cardiac cell apoptosis remains unclear. This study aimed to identify the roles of IGF-II and/or IGF-II receptors (IGF-II/IIR) in cardiomyoblast apoptosis and in hypertensive rat hearts with abdominal aorta ligation. Cultured rat heart-derived H9c2 cardiomyoblasts and excised hearts from Sprague-Dawley rats with 0- to 20-day complete abdominal aorta ligation, a model of ANG II elevation and hypertension, were used. IGF-II/IIR expression, caspase activity, DNA fragmentation, and apoptotic cells were measured by RT-PCR, Western blot, agarose gel electrophoresis, and TUNEL assay following various combinations of ANG II, IGF-II/IIR antibody, CsA (calcineurin inhibitor), SP-600125 (JNK inhibitor), SB-203580 (p38 inhibitor), U-0126 (MEK inhibitor), or Staurosporine (PKC inhibitor) in H9c2 cells. ANG II-induced DNA fragmentation and TUNEL-positive cells were blocked by IGF-II/IIR antibodies and antisense IGF-II, but not by IGF-II sense. IGF-II-induced apoptosis was blocked by IGF-IIR antibody and CsA. The increased gene expressions of IGF-II and -IIR induced by ANG II were reversed by U-0126 and Sp600125, respectively. Caspase 8 activities induced by ANG II were attenuated by U-0126, SP-600125, and CsA. DNA fragmentation induced by ANG II was totally blocked by SP-600125, and CsA and was attenuated by U-0126. In rats with 0- to 20-day complete abdominal aorta ligation, the increases in IGF-II/IIR levels in the left ventricle were accompanied by hypertension as well as increases in caspase 9 activities and TUNEL-positive cardiac myocytes. ANG II-induced apoptosis was reversed by IGF-II/IIR blockade and coexisted with increased transactivation of IGF-II and -IIR, which are mediated by ERK and JNK pathways, respectively, both of which further contributed to cardiomyoblast apoptosis via calcineurin signaling. The increased cardiac IGF-II, IGF-IIR, caspase 9, and cellular apoptosis were also found in hypertensive rats with abdominal aorta ligation.     

Chronic stress induces adrenal hyperplasia and hypertrophy in a subregion-specific manner

The adrenal gland is an essential stress-responsive organ that is part of both the hypothalamic-pituitary-adrenal axis and the sympatho-adrenomedullary system. Chronic stress exposure commonly increases adrenal weight, but it is not known to what extent this growth is due to cellular hyperplasia or hypertrophy and whether it is subregion specific. Moreover, it is not clear whether increased production of adrenal glucocorticoid after chronic stress is due to increased sensitivity to adrenocorticotropic hormone (ACTH) vs. increased maximal output. The present studies use a 14-day chronic variable stress (CVS) paradigm in adult male rats to assess the effects of chronic stress on adrenal growth and corticosterone steroidogenesis. Exogenous ACTH administration (0-895 ng/100 g body wt) to dexamethasone-blocked rats demonstrated that CVS increased maximal plasma and adrenal corticosterone responses to ACTH without affecting sensitivity. This enhanced function was associated with increased adrenal weight, DNA and RNA content, and RNA/DNA ratio after CVS, suggesting that both cellular hyperplasia and hypertrophy occurred. Unbiased stereological counting of cells labeled for Ki67 (cell division marker) or 4,6-diamidino-2-phenylindole (nuclear marker), combined with zone specific markers, showed that CVS induced hyperplasia in the outer zona fasciculata, hypertrophy in the inner zona fasciculata and medulla, and reduced cell size in the zona glomerulosa. Collectively, these results demonstrate that increased adrenal weight after CVS is due to hyperplasia and hypertrophy that occur in specific adrenal subregions and is associated with increased maximal corticosterone responses to ACTH. These chronic stress-induced changes in adrenal growth and function may have implications for patients with stress-related disorders.     

Effect of stress on postnatal growth in weight of rat body and adrenal gland

Seven-week male Wistar rats weighing 145-155 g were housed 10 (control) or 20 (crowded) per cage (50 x 35 x 15 cm). Groups of control and crowded animals were decapitated within 0.5, 1, 3, 7, 14, 21, 28, 35, 56, 70, and 98 days of experiment. The keeping of rats in crowding conditions induced chronic stress, which developed, according to phase changes in plasma concentration of corticosterone and adrenal content of cholesterol, through the stages alarm, resistance, and exhaustion. The postnatal growth of body weight in the stressed rats was behind that in the control. Under the crowding conditions, the adrenal weight did not undergo significant increase, since the stress-induced hypertrophy of the zona fasciculata was compensated by the proportional suppression of the postnatal growth of the zona reticularis. After 56 days, the postnatal growth of the hypertrophied fascicular zone was delayed in the experimental rats.     

Catecholamine effects on cardiac remodelling,oxidative stress and fibrosis in experimental heart failure

The aim of the study was to assess the relationships between oxidative stress, cardiac remodelling and fibrosis on an experimental model of heart failure with adrenergic stimulation. Large myocardial infarction (approximately 50% of the left ventricle myocardium) was obtained by ligation of the left coronary artery of normotensive male Wistar rats. Sham animals were submitted to left thoracotomy without coronary ligation. In order to perform cardiac stimulation by catecholamines, mini-osmotic pumps were implanted in animals 10 weeks after surgery to deliver noradrenalin for a 2-week period. At the end of this period, the following investigations were performed: haemodynamics, morphometry, fibrosis quantification, plasma and tissue catecholamine assay and oxidative stress status. Coronary ligation induced dilatation of left ventricle with compensatory hypertrophy of the right ventricle and of the remaining left ventricle myocardium. This remodelling process was associated in non-infarcted myocardium with increased collagen infiltration and increased oxidative stress. Ten weeks after surgery, the chronic administration of noradrenalin for 2 weeks did not increase oxidative stress. Noradrenalin, however, induced inotropic stimulation and myocardial hypertrophy, but to a lesser extent in infarcted rats compared to sham rats. Our results suggest that noradrenalin infusion to levels in excess of those seen post-infarction is associated with fibrosis and oxidative stress. Moreover, noradrenalin in infarcted animals caused additional fibrosis without further increasing oxidative stress. The mechanism of catecholamine-induced fibrosis may thus involve different processes such as ischaemia, increased mechanical stress, cytokines and neurohormones.     

内源性一氧化氮在高血压心肌肥厚中的作用

目的和方法：本实验用Ｌ精氨酸和一氧化氮合酶（ＮＯＳ）抑制剂ＬＮＡＭＥ观察内源性一氧化氮（ＮＯ）在高血压性心肌肥厚中的作用。结果：腹主动脉缩窄引起大鼠动脉血压显著升高,左心室重量／体重比值显著增加,左心室ＮＯ含量显著下降;Ｌ精氨酸不影响主动脉缩窄大鼠动脉血压,但减轻左心室重量／体重比值,明显升高左心室ＮＯ含量,加入ＬＮＡＭＥ可消除Ｌ精氨酸的上述作用;主动脉缩窄大鼠给予ＬＮＡＭＥ,动脉血压和左心室／体重比值并没有进一步增加;假手术大鼠给予ＬＮＡＭＥ,血压明显升高,左心室重量／体重比值轻度增加;主动脉缩窄大鼠不论是服用Ｌ精氨酸还是ＬＮＡＭＥ,左心室ｃＧＭＰ含量都明显增加。结论：口服Ｌ精氨酸可减轻主动脉缩窄大鼠心肌肥厚但不影响动脉血压,此作用可能是通过Ｌ精氨酸ＮＯ途径实现的,与ｃＧＭＰ机制无关。     

Effect of liposomes on lipid peroxidation and total phospholipids in rabbit ischemic spinal cord model

The effect of spinal cord ischemia (induced by abdominal aorta ligation for 20 minutes) on lipid peroxidation and TPL composition was investigated and discussed in our previous articles. It is known, that partially reduced species of oxygen can be formed under aerobic conditions. For that reason, the effect of ligation release for 60 minutes was observed in experimental animals treated with the selected liposomes. Administration of CP, (CP+SA) and (CP+Chol) liposomes applied 30 minutes before 20 minutes ischemia revealed an ameliorating effect on in vivo and in vitro Fe-dependent peroxidation manifested by TBA-RS accumulation. Combined use of (CP+SA) liposomes with lipophylic form of stobadine (DP 1031) was not more effective. Application of CP liposomes directly before the ligation release slightly increased the antiradical capacity in spinal cord homogenates comparing with not-treated animals. Accumulation of TBA-RS was accompanied by TPL degradation during recirculation period but values of TPL after liposomal treatment were unaffected.     

Decreased density of endothelin binding sites in adrenal glands but not in aorta,of long term infarcted rats

Endothelins (Et-s) are biologically active peptides which play a physiological and pathological role in the cardiovascular regulation. The aim of our study was to verify, in a model of experimental long term myocardial ischemia (15 weeks) in rats, whether there was a modification in the ET binding sites of aorta and adrenal glands. Additionally, Ang II binding sites in adrenal glands were studied. The principal finding of the present study was the down-regulation of ET binding sites in adrenal glands of chronic infarcted rats, whereas no modification of binding parameters for Et-l, in thoractic aorta, nor for Ang II, in adrenal glands, were found.     

Effect of berberine on catecholamine levels in rats with experimental cardiac hypertrophy

The aim of this study is to investigate the effect of berberine on catecholamine level (adrenaline and noradrenaline) in rats with experimental cardiac hypertrophy. Cardiac hypertrophy(CH) was induced by suprarenal abdominal aorta constriction, and the drugs were administered for 8 weeks starting from 4 weeks after surgery. The degree of cardiac hypertrophy was determined by heart and left ventricular weight. The level of adrenaline(AD) and noradrenaline(NA) was detected by HPLC. The data showed that in the CH model rats, the level of plasma and left ventricular tissue AD, and the level of NA in plasma were higher than that of the age-matched controls(indicating increased "total" sympathetic activity). The level of NA in left ventricular tissue of CH model rats was however lower than the age-matched controls. Berberine and captopril showed significant effect on inhibiting the development of cardiac hypertrophy. Berberine decreased plasma NA level and the AD level both in plasma and left ventricular tissue, but had no effect on improving the cardiac NA depletion. Captopril showed significant effect on increasing the depleted cardiac NA and in reducing the elevated plasma NA level. These findings show the efficacy of berberine on modulating the sympathetic nervous activity of rats with experimental cardiac hypertrophy, and reflect the therapeutic potentials of berberine in patients with cardiac hypertrophy and chronic heart failure.     

Reduction of ventricular hypertrophy and fibrosis in spontaneously hypertensive rats by L-arginine

The purpose of this experiment was to explore long-term L-arginine administration on ventricular hypertrophy and cardiac fibrosis in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Twenty-four rats of each strain at eight wks of age were divided into two groups--one receiving L-arginine and the other vehicle for twelve wks. Arterial pressure (AP) and heart rate were monitored. At 20 wks of age, the rats' rings of thoracic aorta were isolated to record isometric tension. The study measured left ventricular weight (LVW), body weight (BW), left ventricular (LV) contents of cGMP, and collagen volume fraction (LVCVF). Histological examination of the LV tissue determined changes in cardiomyocytes. Administration of L-arginine did not alter the AP change in SHR, but reduced the AP in WKY after six wks. Our results showed a significantly higher LVW/BW ratio and LVCVF in vehicle-treated SHR compared to levels in corresponding WKY, whereas, the LV cGMP and nitrite/nitrate measurements were higher in vehicle-treated WKY than in SHR. L-Arginine treatment decreased LVW/BW ratio and LVCVF, while increasing the levels of LV cGMP and nitrite/nitrate only in SHR, consistent with histopathological examinations that showed L-arginine prevented cardiomyocytes from thickness and hypertrophy. Our results suggested that the mechanism of reduction in ventricular hypertrophy and fibrosis following long-term L-arginine administration in SHR may stem from increased myocardial nitric oxide-cGMP signaling, independent of AP and EDV of thoracic aorta.     

Temporal alterations in cardiac fibroblast function following induction of pressure overload

Increases in cardiovascular load (pressure overload) are known to elicit ventricular remodeling including cardiomyocyte hypertrophy and interstitial fibrosis. While numerous studies have focused on the mechanisms of myocyte hypertrophy, comparatively little is known regarding the response of the interstitial fibroblasts to increased cardiovascular load. Fibroblasts are the most numerous cell type in the mammalian myocardium and have long been recognized as producing the majority of the myocardial extracellular matrix. It is only now becoming appreciated that other aspects of fibroblast behavior are important to overall cardiac function. The present studies were performed to examine the temporal alterations in fibroblast activity in response to increased cardiovascular load. Rat myocardial fibroblasts were isolated at specific time-points (3, 7, 14, and 28 days) after induction of pressure overload by abdominal aortic constriction. Bioassays were performed to measure specific parameters of fibroblast function including remodeling and contraction of 3-dimensional collagen gels, migration, and proliferation. In addition, the expression of extracellular matrix receptors of the integrin family was examined. Myocardial hypertrophy and fibrosis were evident within 7 days after constriction of the abdominal aorta. Collagen gel contraction, migration, and proliferation were enhanced in fibroblasts from pressure-overloaded animals compared to fibroblasts from sham animals. Differences in fibroblast function and protein expression were evident within 7 days of aortic constriction, concurrent with the onset of hypertrophy and fibrosis of the intact myocardium. These data provide further support for the idea that rapid and dynamic changes in fibroblast phenotype accompany and contribute to the progression of cardiovascular disease.