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Background/Aims: Interference with endothelial progenitor cell (EPC) neovascularization is a novel therapeutic target for neovascular-related diseases. Angiotensin Ⅱ (Ang Ⅱ) was found to enhance new vessel formation and aggravated neovascular-related diseases. In this study, we investigated the effects of Ang Ⅱ on EPC neovascular-related functions and explored the underlying mechanisms. Methods: EPCs were cultured from bone marrow derived mononuclear cells. The effects of Ang Ⅱ on EPC proliferation, adhesion, migration, and in vitro tube formation were investigated using the MTT assay, adhesion assay, transwell chamber assay, and in vitro tube formation assay respectively. The underlying mechanisms were explored using Western blotting assay. Results: EPC adhesion, migration and in vitro tube formation were promoted by Ang Ⅱ, and the effects were reversed by RhoA/Rho-associated kinases (ROCK) signaling pathway inhibitors including C3 exoenzyme, GGTI-286 and Y-27632. The active form of RhoA was up-regulated by Ang Ⅱ and this effect was abolished by C3 exoenzyme. Moreover, RhoA silencing resulted in a notable inhibition of EPC adhesion, migration and in vitro tube formation, suggesting that RhoA activation played a pivotal role in Ang Ⅱ angiogenic effect. The results also demonstrated that phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun-NH2 kinase was elevated by Ang Ⅱ and attenuated by C3 exoenzyme, GGTI-286 and Y-27632. The enhancing effects of Ang Ⅱ on EPC adhesion, migration and in vitro vasculogenesis were reversed by p38 inhibitor SB202190 and JNK inhibitor SP600125. Conclusion: Ang Ⅱ may enhance EPC neovascular-related functions through activating RhoA/ ROCK and MAPK signaling pathway.

Reactive oxygen species (ROS), originating predominantly from vascular smooth muscle cells (VSMCs), lead to vascular damage and endothelial dysfunction in rats with hypertension. The downstream signaling pathways of farnesyl pyrophosphate (FPP) synthase, Ras-related C3 botulinum toxin substrate 1 (Rac1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, mediate the generation of ROS. The present study investigated the effect of the FPP synthase inhibitor, ibandronate, on ROS production, the possible beneficial effect on endothelial dysfunction and the underlying mechanisms in spontaneously hypertensive rats (SHRs). The SHRs were treated with ibandronate for 30 days. Endothelium‑dependent and independent vasorelaxation were measured in isolated aortic rings. Additionally, VSMCs from the SHRs and Wistar‑Kyoto (WKY) rats were cultured. The production of ROS and activation of NADPH oxidase were determined using fluorescence and chemiluminescence, respectively, in vivo and in vitro. Angiotensin II (Ang II) increased ROS production in the cultured VSMCs from the WKY rats and SHRs, in a concentration‑dependent manner. The Ang II‑induced responses were more marked in the SHR VSMCs, compare with those in the WKY VSMCs, however, the response decreased significantly following ibandronate pretreatment. Treatment with ibandronate significantly decreased the production of ROS, translocation of NADPH oxidase subunit p47phox, and activities of NADPH oxidase and Rac1 in the aorta and VSMCs, and improved the impaired endothelium‑dependent vasodilation in the SHRs. Adding geranylgeraniol, but not farnesol or mevalonate, reversed the inhibitory effects of ibandronate. In addition, inhibiting geranylgeranyl-transferase mimicked the effect of ibandronate on the excess oxidative response. Ibandronate exerted cellular antioxidant effects through the Rac1/NADPH oxidase pathway. These effects may have contributed to the vasoprotective effects on the impaired endothelium in SHRs.

Qiliqiangxin (QL), a traditional Chinese medicine, has been shown to be beneficial for chronic heart failure. However, whether QL can also improve endothelial cell function in diabetic rats remains unknown. Here, we investigated the effect of QL treatment on endothelial dysfunction by comparing the effect of QL to that of benazepril (Ben) in diabetic Sprague-Dawley rats for 8 weeks. Cardiac function was evaluated by echocardiography and catheterization. Assays for acetylcholine-induced, endothelium-dependent relaxation (EDR), sodium nitroprusside-induced endothelium-independent relaxation, serum nitric oxide (NO), and nitric oxide synthase (NOS) as well as histological analyses were performed to assess endothelial function. Diabetic rats showed significantly inhibited cardiac function and EDR, decreased expression of serum NO and phosphorylation at Ser1177 on endothelial NOS (eNOS), and impaired endothelial integrity after 8 weeks. Chronic treatment for 8 weeks with either QL or Ben prevented the inhibition of cardiac function and EDR and the decrease in serum NO and eNOS phosphorylation caused by diabetes. Moreover, either QL or Ben suppressed inducible NOS (iNOS) protein levels as well as endothelial necrosis compared with the diabetic rats. Additionally, QL prevented the increase in angiotensin-converting enzyme 1 and angiotensin II receptor type 1 in diabetes. Thus, chronic administration of QL improved serum NO production, EDR, and endothelial integrity in diabetic rat aortas, possibly through balancing eNOS and iNOS activity and decreasing renin–angiotensin system expression.

Backgrounds: Autophagy is an important process in the pathogenesis of diabetes and plays a critical role in maintaining cellular homeostasis. However, the autophagic response and its mechanism in diabetic vascular endothelium remain unclear. Methods and Results: We studied high-glucose-induced renin-angiotensin system (RAS)-mitochondrial damage and its effect on endothelial cells. With regard to therapeutics, we investigated the beneficial effect of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin II type 1 receptor blockers (ARBs) against high-glucose-induced endothelial responses. High glucose activated RAS, enhanced mitochondrial damage and increased senescence, apoptosis and autophagic-responses in endothelial cells, and these effects were mimicked by using angiotensin II (Ang). The use of an ACEI or ARB, however, inhibited the negative effects of high glucose. Direct mitochondrial injury caused by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) resulted in similar negative effects of high glucose or Ang and abrogated the protective effects of an ACEI or ARB. Additionally, by impairing autophagy, high-glucose-induced senescence and apoptosis were accelerated and the ACEI- or ARB-mediated beneficial effects were abolished. Furthermore, increases in FragEL™ DNA Fragmentation (TUNEL)-positive cells, β-galactosidase activation and the expression of autophagic biomarkers were revealed in diabetic patients and rats, and the treatment with an ACEI or ARB decreased these responses. Conclusions: These data suggest that autophagy protects against senescence and apoptosis via RAS-mitochondria in high-glucose-induced endothelial cells.

Background: Abnormalities of the mevalonate pathway, an important cellular metabolic pathway, are common in many diseases including cardiovascular disease. The mevalonate pathway related enzyme expressions in pressure overload-induced cardiac hypertrophy and associated diastolic dysfunction remains largely unknown. This study aims to investigate whether the expression of mevalonate pathway related enzyme is altered during the progression of cardiac hypertrophy and associated diastolic dysfunction induced by pressure overload. Methods: Male Sprague-Dawley (SD) rats were randomly divided into two groups: the suprarenal abdominal aortic coarctation (AAC) group and the sham group. Results: Histological and echocardiographic assessments showed that there was a significant cardiovascular remodeling in the AAC group compared with the sham group after 3 weeks post-operatively, and the left ventricular (LV) diastolic function was reduced at 8 and 14 weeks post-operatively in the AAC group, without any change in systolic function during the study. The tissue of the heart and the abdominal aorta proximal to the coarctation showed over-expression of several enzymes, including 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FDPS), farnesyltransferase-α (FNTA), farnesyltransferase-β (FNTB), geranylgeranyltransferase type I (GGTase-I) and the activation of their downstream proteins was enhanced. Conclusions: AAC induced compensatory LV hypertrophy to decompensatory diastolic dysfunction, accompanied by altered expression of several key enzymes in the mevalonate pathway.

Farnesyltransferase (FT), an essential enzyme at the downstream of mevalonate pathway, was reported to be upregulated in hypertrophic cardiomyocytes of spontaneously hypertensive rats (SHRs) compared with myocardium of Wistar-Kyoto rats (WKYs). This upregulation was accompanied with cardiac remodeling. This study was designed to determine whether FT inhibition can alter cardiac remodeling in SHRs.Twelve-week-old SHRs were randomized to receive infusion of either NS or FTI-276 (307 μg/kg/d i.v. each n=10). WKY rats served as normal controls (n=6). Echocardiography was performed before and after intervention. SHR hearts were perfused ex vivo for the evaluation of cardiac performance, collagen deposition and biochemical changes (activation of Ras, extracellular-signal regulated kinases/ERK1/2, procollagen type І/Ш, TGF-β1, connective tissue growth factor/CTGF, and bone morphogenetic protein-7/BMP-7 expression).FTI-276 intervention decreased interventricular septum wall thickness at end- diastole (IVSd) and relative wall thickness (RWT) of SHRs (P0.05). Three week intervention with FTI-276 attenuated hydroxyproline content (P0.05), collagen deposition (P0.01), Ras activation, ERK1/2 phosphorylation (P0.01) and mRNA expression of procollagen type I, TGF-β1 and CTGF and elevated mRNA expression of BMP-7 (P0.05) in left ventricle of SHRs.The present study indicated that FT inhibition could attenuate myocardial fibrosis and partly improve cardiac remodeling in SHRs. The beneficial effects might be mediated through suppression of the activation of Ras and ERK1/2 phosphorylation pathway. The enhanced mRNA expression of BMP-7 with inhibition of TGF-β1 and CTGF mRNA expression might be an important mechanism.

The Rho guanosine triphosphatases (Rho GTPases) family, including RhoA, plays an important role in angiotensin II (Ang II)-mediated cardiac hypertrophy. Farnesylpyrophosphate synthase (FPPS)-catalyzed isoprenoid intermediates are vital for activation of RhoA. The present study was designed to investigate the role of FPPS in myocardial hypertrophy mediated with Ang II. First, we demonstrated that FPPS expression was elevated both in cultured neonatal cardiomyocytes (NCMs) following Ang II treatment and in the hypertrophic myocardium of 18-week-old spontaneously hypertensive rats (SHRs). Then, the importance of FPPS was assessed by RNA interference (RNAi) against FPPS in NCMs. Successful FPPS silencing in NCMs completely inhibited the hypertrophy marker genes of β-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP), as well as cell surface area. Furthermore, FPPS knockdown prevented elevated RhoA activity compared with non-silenced controls. Similarly, increased-phosphorylation of p-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) by Ang II was attenuated. In vivo gene transfer also attenuated hypertrophic responses as indexed by left ventricular weight/body weight (LVW/BW), heart weight/body weight (HW/BW), and echocardiography, as well as expression of β-MHC and BNP mRNA in SHRs. In conclusion, FPPS with RhoA associated p-38 and JNK MAPK signaling might play an important role in Ang II-induced cardiac hypertrophy.

Farnesyl pyrophosphate synthase (FPPS, EC 2.5.1.10), an essential enzyme in the mevalonate pathway, catalyzes the synthesis of isoprenoid intermediates. The isoprenoid intermediates are needed for protein isoprenylation of RhoA for its function on regulation of endothelial nitric oxide synthase (eNOS). We previously reported that FPPS were upregulated in spontaneously hypertensive rats (SHR) when compared with Wistar-Kyoto rats (WKY), and this was accompanied by development of endothelial dysfunction. Five-week-old male rats were daily gavaged with vehicle or an FPPS inhibitor (alendronate, 1 or 10mg/kg). After 12-week administration of alendronate, endothelium-dependent and -independent vasorelaxation were measured in isolated aortic rings. Twelve-week of alendronate (10mg/kg/day) treatment restored the impaired endothelium-dependent vasodilation in SHR. Furthermore, long-term treatment with an FPPS inhibitor significantly suppressed RhoA activation and increased phospho-eNOS/eNOS ratio. In conclusion, chronic treatment with an FPPS inhibitor improves the endothelial function in SHR, and the upregulation of phospho-eNOS/eNOS ratio with inhibition of RhoA activation may be an important mechanism.

Sodium aescinate (SA) is used as a vasoactive drug in clinical treatment. This study was designed to investigate the effects of SA on rat isolated thoracic aorta and the possible mechanisms.Isometric tension was recorded in response to drugs in organ bath.The effects of SA obeyed an all-or-nothing response. SA in relatively low dose (or = 50 microg/ml) had an endothelium-independent contractile effect in rat aorta (P0.01), which depended on extracellular Ca(2+) influx via L-type Ca(2+) channel (P0.05). SA in relatively high dose (or = 100 microg/ml) also induced vasoconstriction in Ca(2+)-free medium (P0.01), which was independent of the activity of inositol-1,4,5-trisphosphate receptor (IP(3)R), ryanodine receptor (RYR), and protein kinase C (PKC). SA in relatively high dose (or = 100 microg/ml) dilated both endothelium-intact and endothelium-denuded aortic rings precontracted by phenylephrine (PE) or KCl (each P0.01). SA inhibited extracellular Ca(2+) influx induced by PE or KCl (each P0.01) and had no activation effect on K(+) channels on vascular smooth muscle. The relaxant effect of SA partly depended on the activity of NO synthase but not on the activity of cyclooxygenase.Taken together, this study indicated that SA had dual effects on vascular tension in rat isolated thoracic aorta.

Cyclophosphamide has a role of decreasing high-sensitivity C-reactive protein in the treatment of autoimmune disorders. The effect of cyclophosphasmide on high-sensitivity C-reactive protein was investigated in myocardial ischemia/reperfusion rat.Open-chest rats were submitted to 30 minutes of ischemia and followed for 3, 12, or 24 hours of reperfusion. All 72 rats survived and were divided into sham, ischemia/reperfusion (I/R) and cyclophosphamide groups, and each group included 3 time-point subgroups (3, 12, and 24 hours; n = 8 for each subgroup). Cyclophosphamide (0.75 g/m(2)) or saline was intraperitoneally administrated in the cyclophosphamide or I/R group. A polyethylene tube was inserted into the left ventricular cavity to detect left ventricular systolic pressure, left ventricular end-diastolic pressure, and maximum rate of rise or fall of left ventricular pressure. In the end, blood was collected for detection of high-sensitivity C-reactive protein, and hearts were harvested for histopathologic assessment and infarct size determination.Compared with the I/R group, rats treated with cyclophosphamide showed a significant recovery in myocardial function with improved left ventricular systolic pressure (88.27 +/- 3.78 vs 68.62 +/- 3.78 mm Hg at 3 hours, 92.04 +/- 3.77 vs 63.74 +/- 4.87 mm Hg at 12 hours, and 90.41 +/- 3.98 vs 64.21 +/- 4.88 mm Hg at 24 hours; P.05, respectively). Left ventricular end-diastolic pressure and maximum rate of rise or fall of left ventricular pressure also had similar trends. Infarct size was reduced (26.1% +/- 0.4% vs 40.4% +/- 0.4% at 3 hours, 21.6% +/- 0.4% vs 49.9% +/- 0.4% at 12 hours, and 21.6% +/- 0.4% vs 40.0% +/- 0.4% at 24 hours; P.01, respectively). Histopathologic damage score was attenuated (1.83 +/- 0.14 vs 2.17 +/- 0.14 at 3 hours, 2.33 +/- 0.14 vs 3.17 +/- 0.14 at 12 hours, and 2.83 +/- 0.14 vs 3.83 +/- 0.14 at 24 hours; P.01, respectively). Plasma high-sensitivity C-reactive protein concentration was significantly reduced (29.28 +/- 0.51 vs 32.26 +/- 0.51 ng/mL at 3 hours, 29.06 +/- 0.50 vs 31.8 +/- 0.51 ng/mL at 12 hours, and 28.61 +/- 0.51 vs 31.86 +/- 0.51 ng/mL at 24 h; P.01, respectively).Cyclophosphamide protects myocardial ischemia/reperfusion injury in the rat with a decrease in plasma concentration of high-sensitivity C-reactive protein.

Polysaccharide sulfate (PSS) is a new type of heparinoid synthesized with alginic acid as the basic material and then by chemical introduction of effective groups. Although PSS is successfully applied in ischemic cardio-cerebrovascular disease, its effect on cardiac function after ischemia/reperfusion (I/R) injury has previously not been investigated. The aim of the present study was to investigate whether PSS can protect the heart from I/R injury and the underlying mechanism of protection.Isolated rat hearts were perfused (Langendorff) and subjected to 20 min global ischemia followed by 60 min reperfusion with Kreb's Henseleit solution or PSS (0.3-100 mg/L). Myocardial contractile function was continuously recorded. Creatine kinase (CK) and lactate dehydrogenase (LDH) leakage were measured. Tumor necrosis factor-alpha (TNF-alpha) expression in cardiomyocytes was investigated. Western blot analysis for extracellular regulated kinases (ERKs), c-jun amino-terminal kinase (JNKs) and p38 mitogen-activated protein kinase (MAPK) activity was performed.After I/R, cardiac contractility decreased, CK and LDH levels increased in the coronary effluent, and TNF-alpha expression increased in cardiomyocytes. PSS administration at concentrations of 1-30 mg/L improved cardiac contractility, reduced CK and LDH release and inhibited TNF-alpha production. Phosphorylated-p38MAPK (p-p38MAPK) and p-p54/p46-JNK increased in I/R rat hearts but diminished in PSS (1-30 mg/L) treated hearts. P-p44/p42-ERK levels were unchanged. In contrast, high concentrations of PSS (100 mg/L) had adverse effects that caused a worsening of heart function.PSS has dose-dependent cardioprotective effects on the rat heart after I/R injury. The beneficial effects may be mediated through normalization of the activity of p38 MAPK and JNK pathways as well as controlling the level of TNF-alpha expression.

Objectives:The effect of low dose of cyclophosphamide (CP)protecting cardiacfunction from ischemia-reperfusioninjury was studied on rats. The premise is that CP inhibits immune and inflammatory process, thereby limits I/R injury and improves myocardial function. Methods: Open chest rats were submitted to 30 min of ischemia followed by 3 h, 12 h or 24 h of reperfusion. Rats were divided into sham group, I/R group and CP group, and each group included 3 time-point subgroups (3 h, 12 h and 24 h; n = 8 for each subgroup). A total of 750 mg/m 2 cyclophosphamide was intraperitoneally administrated in CP group and saline was given to I/R group. A polyethylene tube was inserted into the left ventricular cavity to detect left ventricular systolic pressure (LVSP) and dp/dtmax. At the end of the experiment, hearts were harvested for histopathological assessment and infarct size determination. Results: Compared with I/R group, rats treated with low dose CP showed a significant recovery in myocardial function with improved LVSP (88 11 vs 69 11 mmHg of 3 h; 92 11 vs 64 14 mmHg of 12 h; 90 11 vs 64 14 mmHg of 24 h; p < 0.01 respectively). The dp/dtmax also had the similar trends. The myocardial infarct size was reduced in CP group compared to that in I/R group; the infiltration of polymorph nuclear leukocytes (PMNs) in myocardium was decreased in CP group. The histopathological damage score was also attenuated. Conclusions: These findings suggest that low dose CP rescues cardiac function from ischemia-reperfusion injury by alleviating histopathological damage in rat myocardium. # 2008 European Association for Cardio-Thoracic Surgery. Published by Elsevier B.V. All rights reserved.

Background: Diminished myocardial sarcoplasmic reticulum Ca2+-ATPase (SERCA) activity and upregulated phospholamban (PLB) level during cardiac dysfunction, had been reported in many studies. Aims: The current study was designed to examine the effects of rAAV-antisense phospholamban (asPLB) gene transfer on cardiac function, SERCA expression and activity, as well as PLB expression and phosphorylation (Pser16-PLB), in a rat myocardial infarction (MI) model. Methods and results: Rat MI model was generated by ligating the left anterior descending coronary artery. Four weeks later, left ventricular ejection fraction (LVEF), left ventricular systolic pressure (LVSP), the maximal rates of increase and decrease in intraventricular pressure (±dp/dtmax) were significantly depressed, and left ventricular end diastolic pressure (LVEDP) was increased. Myocardial PLB was markedly increased while both SERCA activity and Pser16-PLB level were decreased. In rAAV-asPLB transfected rats, rAAV-asPLB, which was injected into the myocardium around the infarction area immediately after the coronary artery ligation, effectively attenuated the depression of cardiac function, significantly inhibited the expression of PLB, restored Pser16-PLB level and enhanced myocardium SERCA activity. Conclusion: rAAV-asPLB transfer in rats with MI effectively prevented the progression of heart failure.

This study was designed to investigate whether atorvastatin has a beneficial effect on left ventricular (LV) remodeling in spontaneously hypertensive rats (SHR), and then explore the underlying mechanisms involved. 12 SHRs were randomized to receive either distilled water (SHR group, n = 6) or atorvastatin (ATV group, n = 6) for 10 weeks. Age-matched Wistar-Kyoto rats (WKY) gavaged by distilled water were used as normal controls (WKY group, n = 6). By using these rats, we observed the effects of atorvastatin on LV hypertrophy and fibrosis, and investigated atorvastatin-induced cell apoptosis and p27 protein expression. In addition, the serum lipid concentration and blood pressure level were also measured in this study. 10 weeks later, a significant decrease in the cardiosomatic ratio, LV weight to body weight ratio and cardiomyocyte transverse diameter, as well as myocardial hydroxyproline and collagen content was observed in the atorvastatin-treated SHR. In addition, atorvastatin increased the positive rate of cell apoptosis and p27 protein expression. A decreased serum lipid concentration and a reduced systolic blood pressure level were also found in the atorvastatin-treated SHR. These findings demonstrated a beneficial effect of atorvastatin on adverse LV remodeling in SHR, and the induction of cell apoptosis and upregulation of p27 protein may serve as the underlying mechanisms of this action.

Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has long been thought to exert its benefits by reducing cholesterol synthesis, and has been shown to significantly reduce cardiovascular events and mortality in patients with or without coronary artery disease. However, it is still unknown whether acute administration of simvastatin beneficially affects the cardiac function prior or during ischemia-reperfusion. The aim of this study is to evaluate the cardioprotective effect of acute simvastatin treatment on isolated rat hearts or isolated ischemia-reperfusion hearts. Hearts were isolated from male Sprague-Dawley rats and attached to a Langendorff apparatus. The isolated hearts with or without ischemia (15 min) and reperfusion (60 min) were perfused with different concentrations of simvastatin. The parameters of cardiac function (such as left ventricular developed pressure [LVDP], +dp/dt max, and -dp/dt max), heart rate, and coronary flow were recorded. Simvastatin (3-30 micromol/l) significantly increased LVDP, +dp/dt max, and -dp/dt max in isolated rat hearts perfused for 60 min. Heart rate was depressed by 30 micromol/l simvastatin and the coronary flow was increased by 10 and 30 micromol/l simvastatin. At a concentration of 100 micromol/l simvastatin, worsening of heart function and subsequent cardiac arrest occurred. Administration of simvastatin (3-30 micromol/l) significantly preserved cardiac function detected by LVDP, +dp/dt max, and -dp/dt max in the isolated ischemia/reperfused (15/60 min) rat hearts. Simvastatin also significantly decreased heart rate at 30 micromol/l, and increased coronary flow at 10 and 30 micromol/l in these rat hearts. However, the protective effect of simvastatin reverted to increased damage at 100 micromol/l. Only 3 micromol/l simvastatin pretreatment before 15/60 min ischemia-reperfusion altered LVDP, +dp/dt max, and -dp/dt max. Both heart rate and coronary flow were unaltered after simvastatin pretreatment. Since simvastatin at a concentration lower than 100 micromol/l exerted beneficial effects on cardiac function in isolated perfused rat hearts, it could be applied just after myocardial ischemia and reperfusion.

The change of mechanical properties of isolated diabetic rat papillary muscle, myocardial ultrastructure and gene expression of sarco/endoplasic reticulum calcium handling proteins in alloxan-induced diabetic rat heart was investigated.Diabetes was induced in male Sprague-Dawley rats (180-220 g) by administering a single tail-vein injection (40 mg/kg) of alloxan. The control group was injected with normal saline. At the end of 2, 4 and 6 weeks after the induction of diabetes, the right ventricular papillary muscles were isolated and perfused with oxygen saturated Tyrode solution for assessment of the contractile function. Light and electron microscopic analysis was used to analyse the myocardial ultrastructure in rats six weeks after induction. Gene expression of calcium handling proteins was measured by reverse transcription-polymerase chain reaction and Western blot.In the diabetic rats, +dT/dtmax and -dT/dtmax were decreased (P0.01), while 1/2 diastolic intervals were longer than control at the end of 4 and 6 weeks (P0.01). Electron microscopic analysis of the myocardium revealed a spectrum of subcellular remodelling which was characterized by damaged myofibrils and mitochondria and dilated swollen sarcoplasmic reticulum. The levels of both mRNA and protein of phospholamban were significantly increased, whereas 1, 4, 5-trisphophate inositol receptor type 2, ryanodine receptor type 2 mRNAs were significantly decreased compared with that in the age-matched 6 weeks control rats. In contrast, levels of both mRNA and protein of sarco/endoplasic reticulum Ca2+ -ATPase were not affected.Contractile dysfunction of papillary muscles and subcellular remodelling exist in alloxan-induced diabetic rat myocardium. Up-regulation of phospholamban and down-regulation of sarco/endoplasic reticulum calcium release channel may be the molecular mechanism.

This study was designed to investigate the effects of the aqueous ethanol extract of Astragalus membranaceus BUNGE (Leguminosae) on rat thoracic aorta. Isometric tension was recorded in response to drugs in organ bath. In endothelium-intact aortic rings, A. membranaceus extract induced a significant dose-dependent relaxation of the rings precontracted by phenylephrine, which could be inhibited by preincubation with L-N(omega)-nitro-arginine methyl ester or methylthioninium chloride. In endothelium-denuded ones, the extract could dose-dependently relax the rings contracted by phenylephrine, not by KCl; and it could also attenuate contractile response to phenylephrine, not to caffeine or phorbol-12,13-diacetate in Ca(2+)-free medium; but it failed to affect the CaCl(2)-induced enhancement of contractile response to phenylephrine in Ca(2+)-free medium. These results indicate that nitric oxide signaling and Ca(2+)-handling pathway are involved in the A. membranaceus extract-induced vasodilatation.

To investigate the structural changes of aorta, and evaluate the effects of atorvastatin on the remodeling of thoracic aorta in spontaneously hypertensive rats (SHR).Twelve eight-week-old SHR were randomized into atorvastatin treated group (ATV group, n=6) and distilled water group (DW group, n=6); Wistar-Kyoto rats (WKY) were used as normal controls. Atorvastatin was administered to ATV group for 10 weeks by gavage in mixture with distilled water (1 ml); the latter two groups were given the same amount of distilled water by gavage for 10 weeks. Systolic blood pressure of caudal artery was examined before and after treatment, and serum concentrations of total cholesterol, triglycerides and HDL-C were measured. Wall thickness, media thickness, medial cross-sectional area and lumen diameter of thoracic aorta were assessed with computed video processing.Systolic blood pressure in ATV group was markedly lower than that in DW group (P0.01). Compared with DW group and WKY group, serum concentrations of total cholesterol, triglycerides and HDL-C in ATV group were significantly lower (P0.01,P0.05). Wall thickness, media thickness, and medial cross-sectional area to lumen ratio in DW group were significantly higher than those in WKY group and ATV group (P0.01,P0.05), but no such difference was found between WKY group and ATV group (P0.05).Vascular structural changes of aorta are due to the alteration of the vessel wall in early stage of SHR. Atorvastatin can markedly improve vascular remodeling.

Lycopene (Ly), the most common type of antioxidant in the majority of diet types, provides tolerance to ischemia/reperfusion injury. However, the underlying mechanism of the protective effects observed following Ly administration remains poorly investigated. The aim of the current study was to investigate whether Ly prevents damage to hypoxia/reoxygenation (HR)‑induced H9C2 myocardioblasts in an autophagy‑dependent manner. The levels of autophagic markers were detected using western blotting, the level of apoptosis was detected using western blotting and flow cytometry. The activation of autophagy was impaired via knockdown of the expression of 'microtubule‑associated protein 1‑light chain 3β (MAP1LC3B)' and 'Beclin 1'. After 16 h hypoxia, followed by 2 h reoxygenation, the expression levels of the microtubule‑associated protein 1A/1B‑light chain 3 (LC3) and Βeclin 1 autophagic biomarkers, and cell viability were reduced, whereas the percentage of apoptotic cells, and the expression levels of the Bax/B‑cell lymphoma 2 (Bcl‑2) and active caspase‑3 apoptotic biomarkers were increased. Pre‑incubation of the cells with different Ly concentrations reversed the HR‑induced inhibition of autophagy and cell viability, and the HR‑induced elevation in apoptotic levels. The induction of autophagy was accompanied by reduced apoptosis, and decreased expression levels of Bax/Bcl‑2 and active caspase‑3. In addition, the impairment of autophagy by silencing the expression of MAP1LC3B and Beclin 1 accelerated HR‑induced H9C2 cell apoptosis and the Ly‑mediated protective effects disappeared. Furthermore, Bax/Bcl‑2 and active caspase‑3 expression levels were increased. Moreover, Ly‑induced autophagy was associated with increased adenosine monophosphate kinase (AMPK) phosphorylation. Suppressed AMPK phosphorylation using compound C terminates Ly‑mediated cytoprotective effects. Ly treatment improves cell viability and reduces apoptosis as a result of the activation of the adaptive autophagic response on HR‑induced H9C2 myocardioblasts. AMPK phosphorylation may be involved in the progression.

Recent studies have revealed the additional beneficial effects of acetylsalicylic acid (aspirin) in the medication of cardiovascular diseases. The small GTPase RhoA as an important signaling factor is implicated in a wide range of cell functions. This study aimed to investigate the regulatory effect of acetylsalicylic acid on RhoA in vascular smooth muscle cells (VSMCs). We found that aspirin at 300 μM suppressed VSMCs proliferation stimulated by LPS, and this inhibitory effect was partially mediated by inhibiting the iNOS/NO pathway. RhoA overexpression was downregulated by aspirin (both 30 and 300 μM) because of enhanced degradation of RhoA protein. The effect of LPS on increasing active RhoA level was significantly attenuated by aspirin (300 μM), which exerted no effect on RhoA translocation. The promoted RhoA phosphorylation under LPS stimulation, coupled with RhoA protein expression, was greatly decreased by aspirin treatment. No effect of aspirin was found on the expression, activation, and phosphorylation of RhoA in VSMCs devoid of inflammatory stimulation. Our investigation indicates that the regulation of RhoA by aspirin in VSMCs under inflammatory stimulus could be a novel mechanism via which aspirin, apart from the COX-dependent action, exerted the multiple beneficial effects.

Both norepinephrine (NE) and connective tissue growth factor (CTGF) contribute to vascular fibrosis during hypertension. Recent studies indicate that farnesyl pyrophosphate synthase (FPPS) plays an important role in cardiac remodeling in hypertension. However, the role of FPPS in NE-induced fibrotic responses and related molecular mechanisms is unknown. Vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) were stimulated with NE. The fibrotic responses were assessed by measuring CTGF, hydroxyproline (hyp), and α-1 procollagen I levels using Western blot, a hydroxyproline test kit, and real-time quantitative PCR assays, respectively. Ras activity was determined by a pull-down assay using a Ras activation assay kit and detected by Western blot. NE dose-dependently increased fibrosis in SHR-VSMCs, and this increase was significantly reduced by ibandronate, an inhibitor of FPPS. The addition of farnesol, but not geranylgeraniol, partially reversed the inhibitory effects of ibandronate. Furthermore, the anti-fibrotic effects of ibandronate could be mimicked by FTI-276 but not by GGTI-286. A pull-down assay showed that ibandronate reduced the NE-induced Ras activation. Moreover, ibandronate inhibited the NE-induced activation of p38, JNK, and ERK1/2. Only SB203580 (specific inhibitor of p38) diminished the NE-induced CTGF production. These results demonstrated that inhibiting FPPS prevents NE-induced fibrotic responses in SHR-VSMCs and that the Ras kinase and p38 pathways were the underlying mechanisms involved in this process.

To study the effect of phospholamban antisense RNA (asPLB) on sarcoplasmic reticulum Ca2+-ATPase activity and cardiac function in rats with diabetes mellitus (DM) mediated by recombinant adeno-associated virus (rAAV) vector.Six weeks after the induction of DM by streptozotocin injected intraperitoneally, the rats were divided into three groups, namely: DM-rAAV-asPLB group, DM-saline group and DM group (control group). The rats in the DM-rAAV-asPLB group were intramyocardially injected with rAAV-asPLB, the rats in the DM-saline group were injected with saline, and those in the control group did not receive any treatment. Six weeks after gene transfer, the expressions of PLB protein and PLB phosphorylation were detected by Western-blot, while the activity of sarcoplasmic reticulum (SR) Ca2+-ATPase and left ventricular function were measured.The PLB protein expression level was significantly higher whereas the PLB phosphorylation, SR Ca2+-ATPase activity and left ventricular function were significantly lower in the DM-saline group than in the control group. No significant difference was found in PLB protein expression level, PLB phosphorylation or SR Ca2+-ATPase activity between the DM-rAAV-asPLB group and the control group. The left ventricular function in the DM-rAAV-asPLB group was poorer than in the control group and was better than in the DM-saline group.rAAV-asPLB can down-regulate PLB protein expression and up-regulate PLB phosphorylation and SR Ca2+-ATPase activity, thus contributing to the improvement of in vivo left ventricular function.

Overproduction of circulating S100A8/A9 occurs in patients following acute myocardial infarction (AMI). It remains unclear whether ischemia insult per se induces S100A8 and S100A9 expression in cardiac myocytes or even whether the cardiac myocytes participate as a source of these proteins. In this study, western blot analysis and quantitative real-time reverse transcription polymerase chain reaction were used to test samples obtained from isolated spontaneously hypertensive rat hearts and Wistar-Kyoto rat hearts subjected to global normothermic ischemia and from neonatal Wistar rat cardiac myocytes undergoing hypoxia. Ischemia did not increase the expression of S100A8 and S100A9 proteins and mRNA in the myocardium either from the spontaneously hypertensive rat hearts or the Wistar-Kyoto rat hearts. In addition, the levels of S100A8 and S100A9 proteins were unchanged in the neonatal rat cardiac myocytes undergoing hypoxia. However, both ischemia and hypoxia activated NF-kappaB in ischemic myocardium and in hypoxic cardiac cells in a time-dependent manner. The results suggest that the increased serum S100A8/A9 concentrations following AMI were not of cardiac myocyte origin.

RhoA has been shown to play a major role in vascular processes and acetylsalicylic acid (aspirin) is known to exert a cytoprotective effect via multiple mechanisms. In the present study, we aimed at investigating the effect of aspirin on RhoA expression under a stress state in rat VSMCs (vascular smooth muscle cells) and the underlying mechanisms. The expression of iNOS (inducible nitric oxide synthase) and iNOS activity as well as NO concentration was significantly promoted by LPS (lipopolysaccharide) accompanying the elevation of RhoA expression, which was blocked by the addition of the iNOS inhibitor L-NIL [L-N6-(1-iminoethyl)lysine dihydrochloride]. Aspirin (30 μM) significantly attenuated the elevation of RhoA, while indomethacin and salicylate had no similar effect. The sGC (soluble guanylate cyclase) inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) showed the same effect as aspirin in down-regulating RhoA but was reversed by the addition of the cGMP analogue 8-Br-PET-cGMP (β-phenyl-1,N2-ethano-8-bromoguanosine 3′,5′-cyclic monophosphorothioate). 8-Br-PET-cGMP solely enhanced the RhoA expression that was abrogated by preincubation with aspirin. Degradation analysis indicated that aspirin enhanced the protein degradation rate of RhoA and GDP-bound RhoA seemed to be more susceptible to aspirin-enhanced degradation compared with the GTP-bound form. Our results indicate that aspirin attenuates the LPS-induced overexpression of RhoA both by inhibiting new synthesis and accelerating protein degradation, which may help elucidate the multiple beneficial effects of aspirin.

Background: The mevalonate pathway is an important metabolic pathway that plays a key role in multiple cellular processes. The aim of this study was to define whether the enzyme expression in mevalonate pathway changes during cardiovascular remodelling in spontaneously hypertensive rats (SHR). Methods and Results: Hearts and thoracic aortas were removed for the study of cardiovascular remodeling in SHR and Wistar-Kyoto rats (WKY). The protein expression of the enzymes in hearts, aortas and livers was analyzed by western blot. The histological measurements showed that the mass and the size of cardiomyocytes, the media thickness and the media cross-sectional area (MCSA) of the thoracic aorta were all increased in SHR since 3 weeks of age. In the heart, there was overexpression of some enzymes, including 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), farnesyl diphosphate synthase (FDPS), and geranylgeranyltransferase type I (GGTase-I), and downregulation of squalene synthetase (SQS) in SHR since 3 weeks of age. In the aorta, besides similar expressions of HMGR, SQS, FDPS and GGTase-I as in the heart, there was upregulation of farnesyltransferase α at 16 and 25 weeks of age and of farnesyltransferase β in 25-weeks-old SHR. Western blot demonstrated overexpression of HMGR and downregulation of SQS in SHR livers at all ages tested. Conclusions: The cardiovascular remodeling of SHR preceded the development of hypertension, and altered expression of several key enzymes in the mevalonate pathway may play a potential pathophysiological role in cardiovascular remodeling. (Circ J 2011; 75: 1409-1417)

To determine if the diagnostic ultrasound and self-made microbubbles could be used to increase gene transfection and expression in cardiac myocytes by means of the ultrasound-mediated microbubbles destruction.The perfluoropropane-exposed sonicated dextrose albumin(PESDA) microbubbles were made and mixed with indicated volume reporter gene encoding beta-galactosidase prior to gene transfection. Gene transfection into the cultured cardiac myocytes was performed by exposure to the various intense diagnostic ultrasound (1.3 MHz) in the presence of the gene-attached microbubbles. The calcium phosphate precipitation gene transfection was carried out alone or in combination with ultrasound-mediated destruction microbubbles. The cells were harvested 48 h after transfection and beta-galactosidase expression was detected by in situ staining and quantitive assay.Cardiac myocytes exposed to ultrasound with PESDA induced significantly increase in gene expression (60-fold compared with naked plasmids transfection, P0.01). Moreover, it was found that the reporter gene expression not only related with ultrasound intension but also with the microbubbles concentration. In combination with calcium phosphate precipitation gene transfection, ultrasound-mediated destruction microbubbles resulted in more intense gene expression even 6 hours after calcium phosphate precipitation gene transfection.The ultrasonic destruction of gene-loaded microbubble is a highly effective gene transfer method, and it not only acts on the gene entry into cells, but also on the intracellular exogenous DNA expression.

To investigate the changes in the expression of four kinds of calcium regulatory proteins mRNA on the isolated ischemia/ reperfusion (IR) hearts.The rat hearts were divided into two groups: control group and IR group which received 45 min exposure to Krebs-Henseleit solution after 15 min zero-flow global ischemia. The indexes of left ventricular function, such as LVDP, +dp/dt(max), -dp/dt(max), and an arrhythmia scoring system were compared between the two groups. The messenger ribonucleic acid (mRNA) amount of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA), phospholamban (PLB), inositol 1,4,5-trisphosphate receptor2 (IP3R2) and ryanodine receptor2 (RyR2) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and normalized to the mRNA levels of beta-actin.In the IR group, LVDP, +dp/dt(max) and -dp/dt(min) of the isolated hearts were depressed and the high rate of arrhythmias occurred during reperfusion. The levels of SERCA, IP3R2, RyR2 mRNA were lower in the IR isolated hearts group than those in the control group, while there was no difference in the level of phospholamban.These data suggest that myocardial ischemia/reperfusion can induce the depression of cardiac performance and an increased risk of arrhythmias, concomitant with the decrease in SERCA, IP3R2, RyR2 mRNA steady state levels.

To explore the effects of atorvastatin on p27 protein expression and cardiomyocytes apoptosis in spontaneously hypertensive rats (SHR).12 eight-week-old SHR were randomized into 2 groups (n = 6): distilled water group (DW group) and atorvastatin treated group (ATV group). The age-matched wistar-kyoto rats (WKY) were used as controls (WKY group). RT-PCR and Western blot were used to detect, respectively, p27 mRNA and protein expression levels. TUNEL technique was used to detect the apoptotic rate of cardiomyocytes. Meanwhile, left ventricular weight and body weight ratio (LVW/BW), and serum lipids levels were examined in this study.After 10 weeks treatment with atorvastatin, (1) serum lipids levels and LVW and LVW/BW ratio in ATV group were markedly decreased compared to DW group; (2) the apoptotic rate of cardiomyocytes in ATV group was much higher than that in DW group; (3) the mRNA and protein levels of p27 expression in ATV group were also increased markedly compared to DW group.Atorvastatin upregulated the expression of p27 at its mRNA and protein level, and also, facilitated cardiomyocytes apoptosis, which, to a certain extent, might be associated with its effect on the regulation of ventricular hypertrophy.

Background: Gap junctions, which serve as intercellular channels that allow the passage of ions and other small molecules between neighboring cells, play an important role in vital functions, including the regulation of cell growth, differentiation, and development. Statins, the 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase inhibitors, have been shown to inhibit the migration and proliferation of smooth muscle cells (SMCs) leading to an antiproliferative effect. Recent studies have shown that statins can reduce gap junction protein connexin43 (Cx43) expression both in vivo and in vitro. However, little work has been done on the effects of statins on gap junctional intercellular communication (GJIC). We hypothesized in this study that lovastatin inhibits vascular smooth muscle cells (VSMCs) migration through the inhibition of the GJIC. Methods: Rat aortic SMCs (RASMCs) were exposed to lovastatin. Vascular smooth muscle cells migration was then assessed with a Transwell migration assay. Gap junctional intercellular communication was determined by using fluorescence recovery after photobleaching (FRAP) analysis, which was performed with a laser-scanning confocal microscope. Results: The migration of the cultured RASMCs were detected by Transwell system. Cell migration was dose-dependently inhibited with lovastatin. Compared with that in the control (110 ± 26), the number of migrated SMCs was significantly reduced to 72 ± 24 (P < .05), 62 ± 18 (P < .01), and 58 ± 19 (P < .01) at the concentration of 0.4, 2, and 10 umol/L, per field. The rate of fluorescence recovery (R) at 5 minutes after photobleaching was adopted as the functional index of GJIC. The R- value of cells exposed to lovastatin 10 umol/L for 48 hours was 24.38% ± 4.84%, whereas the cells in the control group had an R- value of 36.11% ± 10.53%, demonstrating that the GJIC of RASMCs was significantly inhibited by lovastatin (P < .01). Smaller concentrations of lovastatin 0.08 umol/L did not change gap junction coupling (P > .05). Conclusions: These results suggest that lovastatin inhibits migration in a dose-dependent manner by attenuating JIC. Suppression of gap junction function could add another explanation of statin-induced antiproliferative effect.

The RhoA/Rho-kinase (ROCK) pathway is involved in angiotensin (Ang) II-induced cardiac hypertrophy. However, it is still unclear whether inhibition of farnesylpyrophosphate (FPP) synthase can attenuate Ang II-induced hypertrophic responses, and whether it involves the RhoA/ROCK pathway. The anti-hypertrophic effects of inhibition of FPP synthase with alendronate in Ang II-cultured neonatal cardiomyocytes were partially reversed by geranylgeranyol (GGOH) and were mimicked by GGTI-286, a geranylgeranyl transferase-I inhibitor, C3 exoenzyme, an inhibitor of Rho, or Y-27632, an inhibitor of ROCK. Pull-down assay showed alendronate reduced-active RhoA by Ang II was also partially antagonized by GGOH. This study revealed that the inhibition of FPP synthase by alendronate reduces RhoA activation by diminishing geranylgeranylation which prevents Ang II-induced hypertrophic responses in neonatal cardiomyocytes.Structured summaryMINT-7260047: Rhotekin-RBD (uniprotkb:Q9BST9) physically interacts (MI:0915) with Rhoa (uniprotkb:P61589) by pull down (MI:0096)

Aims The normal expression of Ca2+-regulatory protein is critical for efficient myocardial function. The present study tested the hypothesis that rosuvastatin treatment may attenuate left ventricular (LV) remodelling and dysfunction in the failing heart, which may be associated with alterations of Ca2+-regulatory protein. Methods and results We investigated the change of LV remodelling and function in a rat model of cardiac dysfunction due to myocardial infarction (MI) with or without rosuvastatin (10 mg/kg/day) treatment for 10 weeks. The protein expression of sarcoplasmic reticulum Ca2+ ATPase (SERCA)2a, phospholamban (PLB), and phospho-PLB at serine-16 (pSer16-PLB), as well as SERCA activity, interleukin (IL)-6, and IL-10 levels were evaluated. After rosuvastatin treatment, LV remodelling and dysfunction were prevented. Rosuvastatin prevented the decrease of SERCA2a and pSer16-PLB expression, increased SERCA activity, but showed no effect on PLB expression. Furthermore, rosuvastatin reduced the increased IL-6 level and further elevated IL-10 level in the peri-infarct and remote zones of MI. Serum lipid levels remained unchanged. Conclusion Rosuvastatin is effective in preventing LV remodelling and dysfunction in the failing heart. The molecular mechanism may be related to normalization of SERCA2a expression, SERCA activity, and pSer16-PLB levels, as well as through cytokine alterations independent of its lipid-lowering effect.

Cardiovascular remodeling is closely associated with cholesterol and is attenuated by statins. The spontaneously hypertensive rat (SHR) has a low serum cholesterol level and evident cardiovascular remodeling. The aims of the present study were to characterize the effects of atorvastatin on tissue cholesterol content and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase expression and activity in four tissues from SHR: liver, heart, aorta and kidney.SHR and normotensive Wistar-Kyoto rats (WKY) were treated daily with atorvastatin (50 mg/kg) for 8 weeks. Cholesterol levels of serum and tissues (liver, heart, aorta and kidney) were determined by commercial enzymatic methods. Western blot analysis and high performance liquid chromatogram (HPLC) were used to assay the expression and activity of enzyme respectively.Treatment with atorvastatin decreased cholesterol content and HMGCoA reductase expression and activity in all four tissues of SHR. However, in WKY, atorvastatin only altered HMG-CoA reductase in liver, where the protein expression was upregulated but the enzyme activity was decreased.The present study demonstrates that the effects of atorvastatin on tissue cholesterol content and HMG-CoA reductase are strain- and tissue-specific.

To investigate the vasoconstriction effect of Ixeris sonchifolia in rat thoracic aortic rings and the underlying mechanisms.I. sonchifolia 10-160 g x L(-1) was cumulatively added into organ bath to observe the isometric tension of thoracic aortic rings with intact endothelium or denuded endothelium in basal tension, preconstricted by phenylephrine (PE) or potassium chloride (KCl), and thoracic aortic rings with intact endothelium preincubated frist with captopril, phosphoramidon and indomethacin, respectively, then preconstricted by PE and KCl. The response was recorded and expressed by "relative contraction".Cumulative administration of I. sonchifolia 10-160 g x L(-1) did not affect the vasomotion of aortic rings with endothelium or without endothelium in basal tension. Exposure of intact endothelium rings preconstricted by PE or KCl to I. sonchifolia at concentration (20-160 g x L(-1) induced a significant constriction, which was inhibited by preincubation with captopril, but was not inhibited by preincubation with phosphoramidon or indomethacin. Exposure of endothelium-denuded rings preconstricted by PE or KCl to I. sonchifolia at concentration (10 to approximately 160 g x L(-1) did not effect the vasoconstriction.The results indicate that I. sonchifolia (20 to approximately 160 g x L(-1) can contract the rat thoracic aortic rings with endothelium. The effect of contraction may enhance angiotensin converting enzyme activity and promote endothelium to synthesize angiotensin II. It has no relationship to endothelin or thromboxane A2.

Aims This study was designed to investigate the effects of sodium ferulate (SF) on rat isolated thoracic aortas and the possible mechanisms. Main methods Isometric tension was recorded in response to drugs in organ bath. Cytosolic free Ca 2+ concentration ([Ca 2+ ] i ) was measured using Fluo-3 in cultured rat aortic smooth muscle cells (RASMC). Key findings SF (0.1–30 mM) relaxed the isolated aortic rings precontracted with phenylephrine (PE) and high-K + in a concentration-dependent manner with respective pD 2 of 2.7 ± 0.02 and 2.6 ± 0.06. Mechanical removal of endothelium did not significantly modify the SF-induced relaxation. In Ca 2+ -free solution, SF noticeably inhibited extracellular Ca 2+ -induced contraction in high-K + and PE pre-challenged rings, and suppressed the transient contraction induced by PE and caffeine. The vasorelaxant effect of SF was unaffected by various K + channel blockers such as tetraethylammonium, glibenclamide, 4-aminopyridine, and barium chloride. In addition, SF concentration-dependently reduced the contraction induced by phorbol-12-myristate-13-acetate, an activator of protein kinase C (PKC), in the absence of extracellular Ca 2+ , with the pD 2 of 2.9 ± 0.03. In RASMC, SF had no effect on PE- or KCl-induced [Ca 2+ ] i increase either in the presence or in the absence of external Ca 2+ . Significance These results indicate that SF acts directly as a non-selective relaxant to vascular smooth muscle. The direct inhibition of the common pathway after [Ca 2+ ] i increase may account for the SF-induced relaxation in Ca 2+ -dependent contraction, while the blockage of the PKC-mediated contractile mechanism is likely responsible for the SF-induced relaxation in Ca 2+ -independent contraction.

Previous clinical and experimental studies have demonstrated that statins, the inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, can improve left ventricular function in damaged hearts. Also, the normal expression of Ca(2+) regulatory proteins is critical for efficient myocardial function. However, it is still unclear whether the beneficial effect of statins on cardiac function is associated with alterations of Ca(2+) regulatory proteins. In this study, we investigated the effect of atorvastatin on cardiac function in spontaneously hypertensive rats (SHRs), focusing in particular on its impact on the expression of sarcoplasmic reticulum Ca(2+)-adenosine triphosphatase (SERCA2a), phospholamban (PLB) and its phosphorylated form (phosphorylated PLB), all of which are Ca(2+) regulatory proteins in myocardium. SHRs showed decreases in gene expression of SERCA2a and phosphorylated PLB, and reduction in SERCA activity in the left ventricular myocardium, as well as reduced cardiac function, compared to age-matched Wistar Kyoto rats (WKYs). Furthermore, we showed that in SHRs atorvastatin preserved cardiac dysfunction accompanied by positive alterations in calcium regulatory proteins, with up-regulation in expression of SERCA2a and phosphorylated PLB, and with improvement of SERCA activity. Thus, atorvastatin has positive effects on calcium regulatory proteins, which may be one of the mechanisms of the beneficial effect of statins on cardiac function in spontaneously hypertensive rats.

To study the treatment effect of Wenxin Keli on isoproterenol (ISO) induced heart failure in rats.Sixty six-week old male Wistar rats were randomized into six groups. The rats in control group were only receive distilled water every day. The rats in ISO group also received two subcutaneous injections (85 mg x kg(-1)) of ISO, which were separated by a 24 hour interval and began to receive distilled water 2 weeks later every day. The rats in Wenxin Keli and control group were receive Wenxin Keli (9 mg x kg(-1)) every day. The rats in Wenxin Keli and ISO group received two subcutaneous injections (85 mg x kg(-1)) of ISO, which were separated by a 24 hour interval and began to receive Wenxin Keli (9 mg x kg(-1)) 2 weeks later every day. The rats in valsartan and control group were receive valsartan every day. The rats in valsartan and ISO group received two subcutaneous injections (85 mg x kg(-1)) of ISO, which were separated by a 24 hour interval and began to receive valsartan 30 mg x kg(-1) 2 weeks later every day. Echocardiogram measurement in rats were carried out after 4 weeks and 10 weeks feeding medince of hemodynamic measurement and aconitine induced arrhythmia in rats were carried out after 10 weeks.Echocardiogram indicated that left ventricular internal diameter at diastolic phase (LVIDd), left ventricular internal diameter at systolic phase (LVIDs), LV percent fractional shortening (FS) and LV ejection fraction (EF) were decreased in the ISO group. Treatment with valsartan 4 weeks later, FS and EF were increased compared with the ISO group and 10 weeks later, LVIDd, LVIDs, FS, EF were increased. However, treatment with Wenxin Keli 10 weeks later, LVIDs, FS, EF were not changed obviously. Hemodynamic measurement showed that left ventricular end diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), and dp/dt(max) were improved after 10 weeks of treatment with valsartan. The LVEDP was decreased and dp/dt(max), was increased after 10 weeks of treatment with Wenxin Keli. Aconitine induced arrhythmia in rats in Wenxin Keli and control group were less serious than those in control group, aconitine induced arrhythmia in rats in Wenxin Keli and ISO group were less serious than those in ISO group.Wenxin Keli could greatly improve the ISO induced cardiac dysfunction and protect the aconitine-induced arrhythmia in rats.

To investigate the effects of two different dosage of atorvastatin on endothelium protection in spontaneously hypertensive rats (SHR).SHRs (n=18) were randomly divided into three groups (n=6): SHR control group, 50 mg atorvastatin group and 10 mg group. Six male Wistar-Kyoto rats were selected as normal control group (WKY group). All animals were given vehicle once a day by gavage for 10 weeks. Systolic blood pressure (SBP) was measured before and after treatment with atorvastatin every 2 weeks. Plasma NO and vWF were measured by nitrate reductase method and double antibodies ELISA, respectively. Plasma lipids were also measured.SBP in all SHR groups was much higher than that in WKY group before experiment (P0.01). Compared with SHR control group, SBP significantly decreased in 50 mg atorvastatin group at 6, 8 and 10 weeks and in 10 mg atorvastatin group merely at 10 weeks (P0.05 or P0.01). The plasma levels of NO in SHR control group was significantly lower than those in WKY group (P0.01). After 10 weeks, plasma NO levels in 50 mg and 10 mg atorvastatin groups were markedly higher than those in SHR control group (P0.01 or P0.05). The plasma levels of vWF in SHR control group was significantly higher than those in WKY group (P0.01). After 10 weeks, plasma vWF levels in 50 mg and 10 mg atorvastatin groups were markedly lower than those in SHR control group (P0.01). Plasma lipids in 50 mg and 10 mg atorvastatin groups were lower than those in SHR control group (P0.05 or P0.01).Atorvastatin can decrease blood pressure and significantly improve endothelial function in SHR by increasing plasma NO level and decreasing plasma vWF level.

Abnormal intracellular Ca2+ homeostasis in the myocardium has been suggested as the cause of cardiac hypertrophy, and this process can be prevented by the HMG-CoA reductase inhibitors, statins. In the present study, the effect of atorvastatin on left ventricular hypertrophy was investigated, and then whether the underlying mechanism was related to a defect in intracellular Ca2+ homeostasis explored. Twelve spontaneously hypertensive rats (SHR), at 8 weeks old, were used in this study, and received either distilled water or atorvastatin for ten weeks, with age-matched normotensive Wistar-Kyoto rats (WKY) used as controls. RT-PCR and western blotting were used to detect the mRNA and protein expressions of phospholamban (PLB) and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), respectively, and a colorimetric method used to examine the SERCA2a activity. Additionally, cardiac hypertrophic indices, such as the cardiosomatic ratio, left ventricular weight to body weight (LVW/BW) ratio and cardiomyocytes transverse diameter (TDM), together with the systolic blood pressure (SBP) and serum lipids levels were also examined. After ten weeks, significant decreases were observed in both the mRNA and protein expression levels of SERCA2a, as well as its activity, in the hypertrophied hearts of the SHR. The administration of atorvastatin to the same strains of rats effectively inhibited these decreases, and the above cardiac hypertrophic indices, as well as the SBP and serum lipids levels were significantly decreased. However, no significant changes in the expressions of PLB were observed in WKY, SHR and atorvastatin-treated SHR. These findings demonstrated that through regulation of the PLB and SERCA2a levels in the hearts of SHR, atorvastatin can prevent the cardiac hypertrophy caused due to pressure overload, which provides a relatively new insight into the mechanism of atorvastatin in the prevention of cardiac hypertrophy.

Diabetic cardiomyopathy is characterized by reduced cardiac contractility due to direct changes in myocardium function independent of vascular disease. This study is to investigate the alterations of cardiac sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac function in streptozotocin-induced diabetic rats. Diabetes mellitus (DM) was induced in male Wistar rats by intraperitoneal injection of streptozotocin. The activity of myocardium sarcoplasmic reticulum Ca2+ -ATPase and the left ventricular hemodynamic parameters were measured in DM rats 4 weeks, 6 weeks and 8 weeks after streptozotocin was administered. Phospholamban mRNA expression was detected by reverse transcription-polymerase chain reaction, and the protein levels of phospholamban and sarcoplasmic reticulum Ca2+ -ATPase were determined by Western blot. Normal rats served as control group. It was found that in DM rats 4 weeks after streptozotocin injection, the cardiac function, myocardium sarcoplasmic reticulum Ca2+ -ATPase activity, phospholamban mRNA and phospholamban protein were not significantly changed compared with those in the control rats. At 6 and 8 weeks after the streptozotocin injection, DM rats showed a significant decrease in sarcoplasmic reticulum Ca2+ -ATPase activity and cardiac function, as indicated by an increase of LVEDP and a marked depression in LVSP and +/- dP/dtmax. At the same time points, increases in phospholamban mRNA and protein levels were observed in DM rats. Sarcoplasmic reticulum Ca2+ -ATPase protein level showed no significant alterations in all DM rats compared with that in control rats. Our work confirms that sarcoplasmic reticulum Ca2+ -ATPase activity is depressed in rats with streptozotocin-induced DM, which is accompanied by elevated phospholamban protein level thus contribute to the pathogenesis of cardiac dysfunction in diabetic rats.

To explore the effects of tumor necrosis factor alpha (TNFalpha) on the expression of phospholamban (PLB) and sarco (endo) plasmic reticulum Ca(2+)-ATPase (SERCA2a) and concentration of intracellular free calcium in myocardiocytes.The neonatal rat myocardiocytes were randomly divided into 6 groups: treatment with different concentrations of TNFalpha (1,10,30,50,and 70 microg/L, respectively) and without TNFalpha (control). The mRNA and protein expression of PLB and SERCA2a were detected with one-step reverse transcription-polymerase chain reaction and Western blotting. The changes of intracellular free calcium concentration ([Ca2+]i) in cultured single neonatal rat cardiomyocyte were determined with Fluo-3/AM loading by laser scanning confocal microscopy. RESULTS TNFalpha significantly increased the expression of PLB mRNA and protein in a dose-dependent fashion. The ratio of PLB/beta-actin mRNA in myocardiocytes incubated with 10,30,50, and 70 microg/L TNFalpha significantly increased by 66%, 106%, 141%, and 189% compared with control (P0.05), and protein levels significantly increased by 30%, 48%, 73%, and 114% respectively compared with control (P0.001), but there was no significant difference in PLB mRNA expression between the group treated with 1 microg/L TNFalpha and control group. TNFalpha had no effect on the expression of mRNA and protein of SERCA2a. TNFalpha (50 microg/L) incubated with cell for 24 hours diminished delta[Ca2+]i of single neonatal rat cardiomyocyte about 33% stimulated by isoproterenol (P0.01), but had no effect on delta [Ca2+]i of cardiomyocyte without isoproterenol stimulation.TNFalpha can increase the expression of PLB and decrease delta[Ca2+]i in cardiomyocytes, which may be related with its negative inotropic effects on cardiomyocytes.

Objective This study was designed to investigate the effects of Astragalus membranaceus (AM) and its main components, astragalus saponin (ASP), astragalus polysaccharide (APS) and aminobutyric acid (GABA), on homocysteine (Hcy) induced acute impairment of vascular tone and to explore whether the antioxidant mechanism was involved in AM protective effect. Methods Inhibitory effects of Hcy and protective effects of AM and its main components on endothelium-dependent relaxation of aortic rings were determined by isometric tension recordings and nitric oxide signaling was assayed with 125 I-cGMP RIA Kit. Furthermore, generation of reactive oxygen species (ROS) in endothelial cells was detected using 5-(6)- chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H 2 DCF-DA). Results Hcy significantly inhibited endothelium-dependent relaxation to acetylcholine (ACh) in a dose-dependent manner, and decreased cGMP levels increased by ACh in aorta. Furthermore, superoxide dismutase (SOD), AM, and ASP markedly attenuated inhibition of vasorelaxation and downregulation of cGMP level by Hcy, and APS exerted a tendency to reverse both of the depressive responses, while GABA had no similar effects. Additionally, partially impaired relaxation by Hcy was completely blocked due to the presence of N (ω)-nitro- l -arginine-methyl ester ( l -NAME), which could not be further altered by treatment with AM, ASP, APS or GABA. Finally, Hcy significantly increased intracellular ROS levels in endothelial cells as measured by CM-H 2 DCF-DA fluorescence. SOD, AM, ASP, and APS, but not GABA, inhibited Hcy-stimulated ROS generation. Conclusion This study demonstrated that AM and ASP, potently protected endothelium-dependent relaxation against the acute injury from Hcy through nitric oxide regulatory pathways, in which antioxidation played a key role.

To investigate the ultrastructure of myocardium and gene expression of calcium handling proteins in diabetic rat heart.Diabetes was induced in male Sprague-Dawley rats by a single injection of alloxanm (40 mg/kg ) and the rats in control group were injected with normal saline. At the end of 2, 4, 6 weeks after the induction of diabetes, the animals were sacrificed. The expression of calcium handling proteins was detected by reverse transcription-polymerase chain reaction (RT-PCR) and actin mRNA was used as internal standard. Heart tissue at the apex was obtained for light and electron microscope study.At the end of 4 and 6 weeks, cardiosomatic ratio of diabetic rats was higher than that of control. Electron microscopy revealed a spectrum of subcellular remodeling in myocardium which was characterized by damaged myofibrils and mitochondria, dilated and swollen sarcoplasmic reticulum. Expression of phospholamban mRNAs was significantly increased, but 1,4,5-trisphosphate inositol receptor type 2, ryanodine receptor type 2 mRNAs were significantly decreased compared with those in the age-matched control rats. In contrast, the expression of sarco/endoplasmic reticulum Ca(2+)-ATPase mRNAs was not affected.In diabetic rat heart, gene expression of calcium handling proteins was characterized by up-regulation of phospholamban and down-regulation of sarcoplasmic reticulum calcium release channel while electron microscopic analysis of myocardium revealed a spectrum of subcellular remodeling.

The normal expression of Ca2+-handling protein is critical for efficient myocardial function. The present study was designed to test the hypothesis that beta-blocker treatment may attenuate left ventricular (LV) remodeling and cardiac contractile dysfunction in the failing heart, which may be associated with alterations of Ca2+-handling proteinWe investigated the change of LV remodeling and function in a rat model of heart failure due to myocardial infarction (MI) with or without carvedilol (30 mg/kg/d) or metoprolol (60 mg/kg/d) treatment for 6 weeks (n = 9 in the MI plus carvedilol group, and n = 8 in every other group). The expression of messenger RNA and proteins of sarcoplasmic reticulum Ca2+-adenosine triphosphatase (SERCA) and phospholamban in cardiomyocytes of all rats were also measuredThere was significant LV remodeling and cardiac contractile dysfunction in MI rats. The messenger RNA and protein expression of SERCA were down-regulated (p0.01), but the expression of phospholamban messenger RNA and protein were up-regulated (p0.01) in MI rats compared to sham-operated rats. After the treatment with beta-blockers, LV remodeling and function were clearly improved. Carvedilol was better in attenuating the weight of the LV and the relative weight of the right ventricle than metoprolol (p0.05). beta-Blockers restored the low expression of SERCA (p0.05) but showed no effect on phospholamban expression (p0.05). Moreover, carvedilol induced a more significant improvement of SERCA expression than metoprolol (p0.05)Beta-blockers are effective in preventing LV remodeling and cardiac contractile dysfunction in the failing heart. The molecular mechanism may be related to normalization of SERCA expression.

1. The beta-adrenoceptor antagonist carvedilol reverses cardiac dysfunction in the failing heart. A recent study showed that beta-adrenoceptor antagonists indirectly normalize Ca(2+)-regulatory proteins. The relationship between these two phenomena and the suitable dosage of carvedilol remains unclear. 2. We investigated the change in left ventricular (LV) remodelling and function in a rat model of heart failure due to myocardial infarction (MI) with or without carvedilol (30 or 2 mg/kg per day) treatment for 6 weeks. The expression of mRNA and proteins of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLB) in cardiomyocytes was also measured. 3. There was significant LV remodelling and cardiac contractile dysfunction in MI rats. The expression of SERCA mRNA and protein were downregulated (P0.01), but the expression of PLB mRNA and protein were upregulated (P0.01) in MI rats compared with sham-operated rats. After treatment with carvedilol, LV remodelling and cardiac contractile dysfunction were clearly improved. Low-dose carvedilol was better at improving some parameters of LV remodelling and function than the high dose. Carvedilol partially restored the low expression of SERCA (P0.05), but had no effect on PLB expression (P0.05). Moreover, low-dose carvedilol induced a more significant improvement in SERCA expression than did the high dose (P0.05). 4. The results of the present study suggest that carvedilol is effective in improving LV remodelling and cardiac contractile dysfunction after MI. This may be related to the normalization of SERCA expression.

To investigate the effect of simvastatin on the cardiac contractile function and the alteration of gene and protein expression of the sarcoplasmic calcium regulatory proteins, including sarcoplasmic reticulum Ca2+-ATPase (SERCA), phospholamban (PLB), and ryanodine receptor 2 (RyR2) in rat hearts.Langendorff-perfused rat hearts were subjected to 60-min perfusion with different concentrations of simvastatin (1, 3, 10, 30, or 100 microml/L), and the parameters of cardiac function such as left ventricular developed pressure (LVDP), +dp/dtmax, and -dp/dtmax were determined. The cultured neonatal rat ventricular cardiomyocytes were incubated with simvastatin (1, 3, 10, 30, and 100 micromol/L) for 1 h or 24 h. The levels of SERCA, PLB, and RyR2 expression were measured by reverse transcription-polymerase chain reaction and Western blot. Cytotoxic effect of simvastatin on ventricular cardiomyocytes was assessed by the MTT colorimetric assay.LVDP, +dp/dtmax, and -dp/dtmax of hearts were increased significantly after treatment with simvastatin 3, 10, and 30 micromol/L. In simvastatin-treated isolated hearts, the levels of mRNA expression of SERCA and RyR2 were elevated compared with the control (P0.05), while the mRNA expression of PLB did not change. After the cultured neonatal rat ventricular cardiomyocytes were incubated with 3, 10, 30, and 100 mumol/L simvastatin for 1 h, SERCA and RyR2 mRNA expressions of cardiomyocytes rose, but there was no alteration in protein expressions. However, with the elongation of simvastatin treatment to 24 h, the protein expression of SERCA and RyR2 were also elevated. Additionally, simvastatin (1-30 micromol/L) had no influence on cell viability of cultured cardiac myocytes, but simvastatin 100 micromol/L inhibited the cell viability.Simvastatin improved cardiac performance accompanied by the elevation of SERCA and RyR2 gene and protein expression.

To investigate the effect of Astragalus membranaceus(AM) on vascular circles and the underlying mechanisms.The study was performed with the model of isolate rat thoracic aorta rings in organ bath. When the endothelium of rat thoracic aorta was removed,the effect of accumulated AM on aorta rings in resting tension, or pre-constricted with KCl, or pre-constricted with phenylephrine (PE) was observed. And to explove the mechanism, the aorta rings were incubated with Ca(2+)-free medium alone, or Ca(2+)-free medium plus heparin, or propranolol alone before pre-contraction with PE.AM had no significant effects on aorta rings in resting tension or pre-constricted with KCl. When the concentration of AM was cumulated to 10(-1), 3 x 10(-1),10(0), 3 x 10(0) g/L, it caused concentration-dependent relaxation while aorta rings were pre-constricted with PE(3 x 10(-7)mol/L), compared with the control [(90.4 +/-4.2)% compared with (94.7 +/-2.4)%,(86.1 +/-5.0)% compared with (92.6 +/-3.2)%, (82.3 +/-5.9)% compared with (90.4 +/-3.6) %, (78.3 +/-6.0)% compared with (88.1 +/-4.0)%]. This effect was not inhibited by Ca(2+)-free medium or propranolol alone. However, the effect was attenuated by the co-incubation with heparin and Ca(2+)-free medium [without heparin:(76.2+/-4.3)% compared with (92.3 +/-5.9)%, with heparin: (95.3+/-0.5)% compared with (95.1+/-0.6)%].The results indicate that AM can relax the rat thoracic aorta rings without endothelium. The mechanism may include the inhibition of intracellular calcium ions release by the 1,4,5-triphosphate inositol-receptor-dependent pathway in vascular smooth muscle cells.

To construct a recombinant adeno-associated virus (rAAV) vector containing gene encoding phospholamban antisense RNA (asPLB), and analyse its effect on expression of PLB, expression and activity of sarco-endoplasmic reticulum Ca2+-ATPase (SERCA), and the change of intracellular free Ca2+ concentration ([Ca2+]i) in rat cardiomyocytes.The target gene encoding PLB antisense RNA was inserted inversely into the adeno-associated virus plasmid pAAV-MCS digested by corresponding restricted endonuclease enzyme. The recombinant plasmid and pAAV-RC and pHelper were co-transfected into 293 cell. At the same time, a viral production positive control (rAAV-LacZ) and negative control were performed. The recombinant viruses were used to transfect the cultured rat cardiomyocytes. Site beta-Galactosidase staining were performed to observe the transfer efficiency. Reverse transcription-PCR and Western blot were used to determine the mRNA and protein expression of PLB and SERCA. The activity of SERCA and the [Ca2+]i were measured.The rAAV vectors were constructed successfully and were transfected into rat cardiomyocytes effectively. The PLB mRNA and protein expression were reduced in rat cardiomyocytes transfected by rAAV-asPLB compared with controls. The activity of SERCA was increased. In rest state, the level of [Ca2+]i in the rAAV-asPLB transfected group decreased. The level of [Ca2+]i increased when induced by isoproterenol.AAV-asPLB vector was constructed successfully, which disrupted the expression of PLB, enhanced the activity of SERCA, reduced the resting [Ca2+]i, and improved the cardiac function.

To explore the possible mechanism of cyclovirobuxine D (CVB-D) in countering and inducing arrhythmia, by way of studying its electro-physiological effect on ventricular papillary muscles of rats in vitro.The transmembrane potential of rat's isolated right ventricular papillary muscles were recorded using conventional glass micro-electrode technique.(1) CVB-D in concentration of 13.3-63.3 micromol/L, showed prolonging effect on the action potential repolarization time, mainly the action potential duration 50 (APD50), APD70 and APD90, in dose-dependent manner, in concentration of 33.3-63.3 micromol/L, it could inhibit the resting potential, action potential amplitude (APA) and maximum depolarization velocity (Vmax) in dose-dependent manner. (2) CVB-D also showed time-dependent effect, the effect initiated 10 min after 20 micromol/L was perfused in ventricular muscle, the APD50, APD70 and APD90 were potentiated gradually along with prolongation of action time and reached the peak at 30-40 min, without any potentiation thereafter. (3) CVB-D could markedly prolong the effective refractory period (ERP) of action potential, increase the ratio of ERP/APD. (4) CVB-D in concentration of 33.3 micromol/L could induce frequent, multifocal spontaneous arrhythmia in some cells when the action time was longer than 45 min.CVB-D has the action of anti-ventricular arrhythmia, the mechanism is correlated with the prolongation of APD and ERP of ventricular muscle as well as the increase of ERP/APD ratio, while it also has the effect of inducing arrhythmia, the mechanism might be concerned with excessive prolongation of APD and the inhibition on RP, APA and Vmax.

To evaluate the expression of nitric oxide synthase III (NOS III) mRNA in the heart, aorta, kidney and liver of spontaneously hypertensive rats (SHR).Two hundred and ninety-four total RNA samples were obtained from the tissues of ventricle, aortic smooth muscle, kidney and liver of SHR and normotensive rats (Wistar-Kyoto rats, WKY). RNA array was used to determine the mRNA levels of NOS III of the two groups.Compared with WKY, the systolic blood pressure increased significantly in SHR at 6-week-old, 8-week-old, 10-week-old and 12-week-old [(158.50 +/-7.69 vs 108.67 +/-5.89) mmHg, (174.33 +/-4.46 vs 128.50 +/-4.97) mmHg, (198.00 +/-13.45 vs 142.00 +/-3.58) mmHg, (216.67 +/-8.91 vs 141.17 +/-4.92) mmHg, P0.01], and the ventricle/body weight ratio was significant higher at 10-week-old and 12-week-old [(4.08 +/-0.17 vs 3.59 +/-0.11, 4.05 +/-0.18 vs 3.40 +/-0.19)mg/g, P0.01]. In the heart tissue and the kidney, the mRNA levels of NOS III were significantly increased at 6-week-old, 8-week-old, 10-week-old and 12-week-old (1.12 +/-0.18 vs 0.90 +/- 0.15, 1.46 +/- 0.34 vs 1.06 +/-0.18, 1.66 +/- 0.31 vs 1.21 +/- 0.30, 1.98 +/- 0.40 vs 1.31 +/-0.38, P0.05) and at 4-week-old, 6-week-old, 8-week-old, 10-week-old and 12-week-old (1.10 +/- 0.21 vs 0.81 +/-0.11, 1.28 +/-0.18 vs 0.95 +/-0.13,1.31 +/-0.23 vs 0.99 +/-0.23, 1.70 +/-0.30 vs 1.08 +/-0.25, 1.83 +/-0.33 vs 1.15 +/-0.20, P0.05 or P0.01), respectively. There was no significant difference of the NOS III expression in the liver and no significant signals were detected in the aortic smooth muscle.The results provide the evidence of the increased expression of NOS III in different tissues in SHR and suggests the progressive nature of essential hypertension.