Involvement of calcineurin in angiotensin II-induced cardiomyocyte hypertrophy and cardiac fibroblast hyperplasia of rats

A rapidly emerging body of literature implicates a pivotal role for the Ca2+-calmodulin-dependent phosphatase, calcineurin, as a cellular target for a variety of Ca2+-dependent signaling pathways culminating in cardiac hypertrophy. The aim of the present study was to test whether calcineurin is involved in the signal transduction of angiotensin II (AngII)-induced cardiac myocyte hypertrophy and fibroblast hyperplasia. Firstly, we observed that calcineurin activity was significantly increased in AngII-stimulated cardiac myocytes as well as fibroblasts, but was markedly inhibited by Losartan (50 micromol/l), H7 (50 micromol/l), and Fura-2/AM (5 micromol/l). It is indicated that AngII-induced activation of calcineurin is through an ATI receptor, may be dependent on the sustained increases of [Ca2+]i, and be regulated by protein kinase C. In a second experiment, we found that cyclosporin (0.1-10micromol/l), a specific inhibitor of calcineurin, decreased the protein synthesis rate in AngII-stimulated cardiomyocytes and the DNA synthesis rate in AngII-treated fibroblasts in a dose-dependent manner. In the latter experiment, calcineurin inhibition reduced the mRNA level of the atrial natriuretic factor gene. These results indicate that calcineurin is involved in the signal transduction of AngII-induced cardiomyocyte hypertrophy and fibroblast hyperplasia.     

Peroxisome proliferator-activated receptor beta/delta activation improves angiotensin II-induced cardiac hypertrophy in vitro

Agonists of the peroxisome proliferator-activated receptor alpha (PPARalpha) and gamma (gamma) exert anti-proliferative and anti-inflammatory effects that led to the testing of these drugs in experimental cardiac hypertrophy. However, the effect of PPAR beta/delta (beta/delta) agonists in hypertrophy is not yet known. In this paper, an experiment was conducted to explore whether PPARbeta/delta activation has an effect on cardiac hypertrophy. An in vitro cardiomyocyte hypertrophy from neonatal rats was induced with Angiotensin II (Ang II1micromol x L(-1)) stimulation. For the examination of PPAR beta/delta effect, the cultured rat cardiac myocytes were pretreated with GW0742 (10 micromol.L(-1)), an agonist of PPARbeta/delta, for 48h before Ang II stimulation. The following parameters in the cultured cells were determined: surface areas of myocytes were measured by the NIH Image Software; (3)H-leucine incorporation into myocytes was counted by liquid scintillometer; mRNA expression of PPARbeta/delta, ANP, BNP, MMP9, MMP2, and IL-1beta was detected by RT-PCR; PPARbeta/delta protein expression was evaluated with immunofluorescence staining; GW0742 could ameliorate Ang II-induced cardiomyocyte hypertrophy, as indicated by its inhibitory effects on the surface area of myocytes, and ANP and BNP mRNA expressions in myocytes and (3)H-leucine incorporation into myocytes. Meanwhile, GW0742 pretreatment exerted inhibition on mRNA expression augmentation of such cytokines as MMP9, MMP2, and IL-1beta in hypertrophic myocytes. In addition, the down-regulated expression of PPARbeta/delta mRNA and protein in hypertrophic myocytes was also significantly reversed by GW0742. We demonstrate for the first time that GW0742 exerts a beneficial effect on Ang II-induced cardiac hypertrophy and the relation to inflammation response.     

Neurally-mediated increase in calcineurin activity regulates cardiac contractile function in absence of hypertrophy

OBJECTIVE: The calcineurin pathway has been involved in the development of cardiac hypertrophy, yet it remains unknown whether calcineurin activity can be regulated in myocardium independently from hypertrophy and cardiac load. METHODS: To test that hypothesis, we measured calcineurin activity in a rat model of infrarenal aortic constriction (IR), which affects neurohormonal pathways without increasing cardiac afterload. RESULTS: In this model, there was no change in arterial pressure over the 4-week experimental period, and the left ventricle/body weight ratio did not increase. At 2 weeks after IR, calcineurin activity was increased 1.8-fold (P<0.05) and remained elevated at 4 weeks (1.7-fold, P<0.05). Similarly, the cardiac activity of calcium calmodulin kinase II (CaMKII) was increased significantly after IR, which confirms a regulation of Ca(2+)-dependent enzymes in this model. In cardiac myocytes, the increased activity of calcineurin was accompanied by a significant decrease in L-type Ca(2+) channel activity (I(Ca)) and contraction velocity (-dL/dt). Cardiac denervation prevented the activation of calcineurin after IR, which demonstrates that a neurohormonal mechanism is responsible for the changes in enzymatic activity. In addition, cardiac denervation suppressed the effects of IR on I(Ca) and -dL/dt, which shows that calcineurin activation is related to altered contractility. However, action potential duration, the densities of inward rectifier K(+) currents (I(K1)), and outward K(+) currents (I(to) and I(K)) were not altered in IR myocytes. CONCLUSIONS: Calcineurin can be activated in the heart through a neural stimulus, which induces alterations in Ca(2+) currents and contractility. These effects occur in the absence of myocyte hypertrophy, electrophysiological changes in action potential, and K(+) channel currents.     

Nicotine inhibits cardiac apoptosis induced by lipopolysaccharide in rats

OBJECTIVES: Apoptosis develops in several heart diseases, but the therapeutic options are limited. It was hypothesized that nicotine, which inhibits apoptosis in several cells, inhibits cardiac apoptosis induced by lipopolysaccharide (LPS). BACKGROUND: Over-the-counter nicotine produces sustained levels (10 to 25 ng/ml) that may be antiapoptotic. Low levels of LPS induce apoptosis by activating tissue renin-angiotensin to stimulate angiotensin II, type 1 (AT(1)) receptors in cardiac myocytes. METHODS: Adult Sprague Dawley rats were pretreated with nicotine (6 mg/kg/day) or saline for seven to ten days (miniosmotic pumps). The LPS (1 mg/kg) was injected intravenously. Toll-like receptor 4 (TLR4) and angiotensinogen messenger ribonucleic acid (mRNA) were measured in the heart after 0, 4, 8, 16, and 24 h. Cardiac apoptosis was measured by terminal deoxy-nucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining after 24 h. In vitro effects of LPS (10 ng/ml, 24 h) were studied in cardiac myocytes isolated from rats pretreated with nicotine for 7 to 10 days, or after pre-exposing myocytes to nicotine (15 ng/ml) for 1, 4, 16, or 24 h. RESULTS: Neither nicotine nor LPS affected systolic blood pressure. The LPS increased cardiac apoptosis after 24 h in saline-treated, but not nicotine-treated rats, despite similar increases in cardiac TLR4 and angiotensinogen mRNA over 8 to 16 h. The LPS-induced apoptosis was blocked by pre-exposing myocytes to nicotine for 4 to 24 h (partial inhibition after 1 h). Nicotine did not inhibit apoptosis induced by angiotensin II (100 nM, 24 h). CONCLUSIONS: Therapeutic levels of nicotine inhibit LPS-induced cardiac apoptosis. This occurs after LPS increases TLR4 and angiotensinogen mRNA, but proximal to AT(1) receptor activation. Nicotine may be a novel inhibitor of cardiac apoptosis in conditions associated with circulating LPS (e.g., decompensated heart failure, acute and chronic infections).     

Cardiomyocyte calcineurin signaling in subcellular domains: from the sarcolemma to the nucleus and beyond

The serine-threonine phosphatase calcineurin is activated in cardiac myocytes in the diseased heart and induces pathological hypertrophy. Calcineurin activity is mainly triggered by calcium/calmodulin binding but also through calpain mediated cleavage. How controlled calcineurin activation is possible in cardiac myocytes, which typically show a 10-fold difference in cytosolic calcium concentration with every heartbeat, has remained enigmatic. It is now emerging that calcineurin activation and signaling occur in subcellular microdomains, in which it is brought together with target proteins and exceedingly high concentrations of calcium in order to induce downstream signaling. We review current evidence of subcellular calcineurin mainly at the sarcolemma and the nucleus, but also in association with the sarcoplasmic reticulum and mitochondria. We also suggest that knowledge about subcellular signaling could help to develop inhibitors of calcineurin in specific microdomains to avoid side-effects that may arise from complete calcineurin inhibition.     

Heme oxygenase-1 inhibition of MAP kinases, calcineurin/NFAT signaling, and hypertrophy in cardiac myocytes

OBJECTIVE: Heme oxygenases (HO) are the rate-limiting enzymes in heme degradation, catalyzing the breakdown of heme to equimolar quantities of biliverdin (BV), carbon monoxide (CO), and ferrous iron. The inducible HO isoform, HO-1, confers protection against ischemia/reperfusion (I/R)-injury in the heart. We hypothesized that HO-1 and its catalytic by-products constitute an antihypertrophic signaling module in cardiac myocytes. METHODS AND RESULTS: The G protein-coupled receptor (GPCR) agonist endothelin-1 (ET-1) (30 nmol/l) stimulated a robust hypertrophic response in cardiac myocytes isolated from 1- to 3-day-old Sprague-Dawley rats, with increases in cell surface area (planimetry), sarcomere assembly (confocal laser scanning microscopy), and prepro-atrial natriuretic peptide (ANP) mRNA expression. Adenoviral overexpression of HO-1, but not beta-galactosidase, significantly inhibited ET-1 induced cardiac myocyte hypertrophy. The antihypertrophic effects of HO-1 were mimicked by BV (10 micromol/l) and the CO-releasing molecule [Ru(CO)3Cl2]2 (10 micromol/l), strongly suggesting a critical involvement of BV and CO in the antihypertrophic effects of HO-1. Both BV and CO suppressed extracellular signal-regulated kinases (ERK1/ERK2) and p38 mitogen-activated protein kinase (MAPK) activation by ET-1 stimulation. Moreover, BV and CO inhibited the prohypertrophic calcineurin/NFAT pathway. This inhibition occurred upstream from calcineurin because BV and CO inhibited NFAT activation in response to ET-1 stimulation but not in response to adenoviral expression of a constitutively active calcineurin mutant. Upstream-inhibition of the calcineurin/NFAT pathway by CO occurred independent from cGMP and cGMP-dependent protein kinase type I (PKG I). CONCLUSIONS: Heme oxygenase-1 and its catalytic by-products, BV and CO, constitute a novel antihypertrophic signaling pathway in cardiac myocytes. Biliverdin and CO inhibition of MAPKs and calcineurin/NFAT signaling provides a mechanistic framework how heme degradation products may promote their antihypertrophic effects.     

Angiotensin II and serotonin potentiate endothelin-1-induced vascular smooth muscle cell proliferation

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation induced by various growth factors has been implicated in a wide variety of pathological processes, including hypertension, atherosclerosis and restenosis after angioplasty. OBJECTIVES: To investigate the interactions among well-known potent vasoconstrictor substances, endothelin-1 (ET-1), angiotensin II (Ang II), and serotonin (5-HT), on VSMC proliferation. METHODS: Growth-arrested rabbit VSMCs were incubated with different concentrations of ET-1 in the absence or presence of Ang II, 5-HT, or both. VSMC proliferation was examined by increases in incorporation of [3H]thymidine into DNA and in cell number. RESULTS: ET-1, Ang II and 5-HT stimulated DNA synthesis in a dose-dependent manner. ET-1 had a maximal effect at a concentration of 0.5 micromol/l (259% of control), Ang II at 1 micromol/l (173%), and 5-HT at 50 micromol/l (205%). When added together, ET-1 (0.1 micromol/l) and Ang II (1 micromol/l) synergistically induced DNA synthesis (341%). When the vasoconstrictors were tested in combination, even non-mitogenic concentrations of ET-1 (0.01 nmol/l) potentiated 5-HT (5 micromol/l)-induced DNA synthesis (404%). Co-incubation of ET-1 (0.01 micromol/l) with Ang II (1 micromol/l) and 5-HT (5 micromol/l) synergistically induced DNA synthesis (566%). These effects on DNA synthesis were paralleled by an increase in cell number. The ETA/B non-selective receptor antagonist, TAK044 (1 micromol/l) and the ETA receptor antagonist, BQ123 (1 micromol/l), but not the ETB receptor antagonist, BQ788 (1 micromol/l), inhibited the mitogenic effect of ET-1 and its interaction with Ang II or 5-HT. In addition, TAK044 (1 micromol/l) or BQ123 (1 micromol/l) along with the angiotensin II type 1 (AT1) receptor antagonist, candesartan (1 micromol/l), the 5-HT2A receptor antagonist, sarpogrelate (10 micromol/l), or both, inhibited the interactions of ET-1 with Ang II or 5-HT. CONCLUSIONS: Our results suggest that Ang II and 5-HT could potentiate ET-1-induced VSMC proliferation. Inhibition of ETA, AT1, and 5-HT2A may be effective in the treatment of VSMC proliferative disorders associated with hypertension, atherosclerosis and restenosis after angioplasty.     

Mechanical stretch-induced hypertrophy of neonatal rat ventricular myocytes is mediated by beta(1)-integrin-microtubule signaling pathways

BACKGROUND: Mechanical stress plays a crucial role in tissue morphogenesis and remodeling. These processes depend in part on force transmission mediated through integrins and the cytoskeleton. METHODS: Ventricular myocytes isolated from neonatal Sprague-Dawley rats (NRVMs) were exposed to persistent centrifugal force stretch for 12 or 24 h. The NRVMs were exposed to colchicine (4 micromol/ml) and anti-integrin beta1 specific antibody (10 microg/ml). Cell viability was assessed by MTT assay and lactate dehydrogenase (LDH) activity. Incorporation of 3H-leucine, and atrial natriuretic peptide (ANP) and angiotensin II (Ang II) levels were assessed. Pixel intensity and distribution of the microtubule were estimated from laser scanning confocal images. RESULTS: Changes in LDH release and the MTT assay showed that 180 rpm. centrifugal force had minimal effect on the viability and number of NRVMs. Mechanical stretch significantly increased 3H-leucine incorporation into cardiomyocytes. Anti-integrin beta1 blocking antibody effectively inhibited the increase in 3H-leucine incorporation and release of ANP (p < 0.05). Following anti-integrin-beta1-blocking antibody, the pixel intensity of the microtubule image was decreased after both12 and 24 h stretch, this was similar to the effect of colchicine. Both treatments also inhibited the secretion of Ang II induced by stretch (p < 0.05). CONCLUSIONS: Anti-integrin-beta1-blocking antibody and colchicine had similar effects, partly inhibiting the stretch-induced increase in microtubule polymerization and the secretion of Ang II in hypertrophic cardiac myocytes.     

Rac2 GTPase activation by angiotensin II is modulated by Ca2+/calcineurin and mitogen-activated protein kinases in human neutrophils

Angiotensin II (Ang II) highly stimulates superoxide anion production by neutrophils. The G-protein Rac2 modulates the activity of NADPH oxidase in response to various stimuli. Here, we describe that Ang II induced both Rac2 translocation from the cytosol to the plasma membrane and Rac2 GTP-binding activity. Furthermore, Clostridium difficile toxin A, an inhibitor of the Rho-GTPases family Rho, Rac and Cdc42, prevented Ang II-elicited O2-/ROS production, phosphorylation of the mitogen-activated protein kinases (MAPKs) p38, extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase 1/2, and Rac2 activation. Rac2 GTPase inhibition by C. difficile toxin A was accompanied by a robust reduction of the cytosolic Ca(2)(+) elevation induced by Ang II in human neutrophils. Furthermore, SB203580 and PD098059 act as inhibitors of p38MAPK and ERK1/2 respectively, wortmannin, an inhibitor of phosphatidylinositol-3-kinase, and cyclosporin A, a calcineurin inhibitor, hindered both translocation of Rac2 from the cytosol to the plasma membrane and enhancement of Rac2 GTP-binding elicited by Ang II. These results provide evidence that the activation of Rac2 by Ang II is exerted through multiple signalling pathways, involving Ca(2)(+)/calcineurin and protein kinases, the elucidation of which should be insightful in the design of new therapies aimed at reversing the inflammation of vessel walls found in a number of cardiovascular diseases.     

环孢素A对儿茶酚胺诱导的大鼠心肌肥大的作用

目的 观察环孢素A（CaA)对儿茶酚胺诱导的大鼠心肌肥大的作用。方法 雌性Wistar大鼠21只,实验分三组,每组7只：（1）单纯肥大组：给大鼠皮下注射异丙肾上腺素（5mg.kg^-1,d^-1),连续10d:(2)CsA治疗组：除注射异丙肾上腺素外,同时腹腔注射CsA(20mg.kg^-1,d^-1),连续10d;（3）对照组：不作特殊处理。三组大鼠计大小,组织形态,心系数以及心肌组织抑制钙调神经磷酸酶CaN,丝裂素活化蛋白激酶（MAPK）及蛋白激酶C（PKC）活性的变化。在培养的大鼠心肌细胞上,观察CsA对去甲肾上腺素（NE）刺激的^3H－亮氨酸掺入的影响。结果 单纯注射异丙肾上腺素组的大鼠心脏明显增大,心肌细胞肥大,排列紊乱,并出现广泛间质纤维化,CaA治疗组大鼠心脏未明显增大,但仍可见部分心肌纤维化,大鼠心重及心系数明显低于单纯肥大组（P＜0．05）。肥大组大鼠心肌组织CaN活性明显高于对照组（P＜0．05）,CaA治疗组大鼠心肌组织CaN活性低于肥大组（P＜005）,三组大鼠心肌组织MAPK活性差异无显著性,但肥大组大鼠心肌组织PKC活性较对照组增高4倍（P＜0．001）,CsA治疗组的大鼠心肌组织PKC活性较肥大组下降50％（P＜0．001）,CsA可明显抑制NE刺激的大鼠心肌细胞^3H-亮氨酸掺入,结论 CaN信号通中可能以儿茶酚胺诱导的心肌肥大中起一定作用,CsA可阻滞儿茶酚胺诱导的心肌肥大,这种作用可能主要通过抑制CaN及PKC活性,阻断CaN和PKC介导的信号传导通路所致。     

Increased Na+/H+-exchange activity is the cause of increased [Na+]i and underlies disturbed calcium handling in the rabbit pressure and volume overload heart failure model

OBJECTIVE: Cytosolic sodium ([Na+]i) is increased in heart failure (HF). We hypothesize that up-regulation of Na+/H+-exchanger (NHE) in heart failure is causal to the increase of [Na+]i and underlies disturbance of cytosolic calcium ([Ca2+]i) handling. METHODS: Heart failure was induced in rabbits by combined volume and pressure overload. Age-matched animals served as control. [Na+]i, cytosolic calcium [Ca2+]i and cytosolic pH (pH(i)) were measured in isolated left ventricular midmural myocytes with SBFI, indo-1 and SNARF. SR calcium content was measured as the response of [Ca2+]i to rapid cooling (RC). Calcium after-transients were elicited by cessation of rapid stimulation (3 Hz) in the presence of 100 nmol/l noradrenalin. NHE and Na+/K+-ATPase activity were inhibited with 10 micromol/l cariporide and 100 micromol/l ouabain, respectively. RESULTS: At all stimulation rates (0-3 Hz) [Na+]i and diastolic [Ca2+]i were significantly higher in HF than in control. With increasing frequency [Na+]i and diastolic [Ca2+]i progressively increased in HF and control, and the calcium transient amplitude (measured as total calcium released from SR) decreased in HF and increased in control. In HF (at 2 Hz), SR calcium content was reduced by 40% and the calcium gradient across the SR membrane by 60%. Fractional systolic SR calcium release was 90% in HF and 60% in control. In HF the rate of pH(i) recovery following acid loading was much faster at all pH(i) and NHE dependent sodium influx was almost twice as high as in control. In HF cariporide (10 micromol/l, 5 min) reduced [Na+]i and end diastolic [Ca2+]i to almost control values, and reversed the relation between calcium transient amplitude and stimulation rate from negative to positive. It increased SR calcium content and SR membrane gradient and decreased fractional systolic SR depletion to 60%. Cariporide greatly reduced the susceptibility to develop calcium after-transients. In control animals, cariporide had only minor effects on all these parameters. Increase of [Na+]i with ouabain in control myocytes induced abnormal calcium handling as found in HF. CONCLUSIONS: In HF up-regulation of NHE activity is causal to increased [Na+]i and secondarily to disturbed diastolic, systolic and SR calcium handling. Specific inhibition of NHE partly normalized [Na+]i, end diastolic [Ca2+]i, and SR calcium handling and reduced the incidence of calcium after-transients. Chronic treatment with specific NHE inhibitors may provide a useful future therapeutic option in treatment of developing hypertrophy and heart failure.     

Single L-type Ca(2+) channel regulation by cGMP-dependent protein kinase type I in adult cardiomyocytes from PKG I transgenic mice

OBJECTIVE: Calcium entry via the L-type Ca(2+) channel (LTCC) is crucial for excitation-contraction (EC) coupling and activation of Ca(2+)-dependent signal transduction pathways in cardiac myocytes. Both nitric oxide (NO), signaling via cGMP, and acetylcholine, signaling via the muscarinic receptor, have been identified as negative regulators of beta-adrenoreceptor-stimulated LTCC activity in cardiac myocytes. METHODS: To examine the potential role of cGMP-dependent protein kinase type I (PKG I) in the inhibitory effects of NO/cGMP and the muscarinic receptor on LTCC activity, we generated transgenic (TG) mice overexpressing PKG I selectively in cardiac myocytes under the control of the alpha-myocin heavy chain promoter. Single LTCC-gating properties were assessed in isolated ventricular myocytes from adult wild-type (WT) and PKG I transgenic (TG) mice. RESULTS: Basal LTCC activity (peak average current, mean open probability, mean availability) was significantly decreased by the nitric oxide donor DEA-NO (0.1 micromol/l) and the cGMP-analog 8-Br-cGMP (1 mmol/l) in TG but not in WT cardiac myocytes. Conversely, muscarinic (carbachol, 1 micromol/l) stimulation had no significant effect on basal LTCC activity in either WT or TG cardiac myocytes. beta-Adrenergic stimulation with isoproterenol (1 micromol/l) increases single LTCC activity in WT and TG cardiac myocytes to the same extent. The inhibitory effects of DEA-NO and 8-Br-cGMP on isoproterenol activation of the LTCC current were significantly enhanced in TG as compared to WT cardiac myocytes. By contrast, carbachol inhibition of isoproterenol-stimulated single LTCC activity was not enhanced in TG cardiac myocytes. CONCLUSION: Transgenic overexpression of PKG I augments NO/cGMP inhibition but not muscarinic inhibition of single LTCC activity, indicating that PKG I is a downstream target for NO/cGMP, but not the muscarinic receptor in adult cardiac myocytes.     

AT1 blockade prevents glucose-induced cardiac dysfunction in ventricular myocytes: role of the AT1 receptor and NADPH oxidase

Enhanced tissue angiotensin (Ang) II levels have been reported in diabetes and might lead to cardiac dysfunction through oxidative stress. This study examined the effect of blocking the Ang II type 1 (AT1) receptor on high glucose-induced cardiac contractile dysfunction. Rat ventricular myocytes were maintained in normal- (NG, 5.5 mmol/L) or high- (HG, 25.5 mmol/L) glucose medium for 24 hours. Mechanical and intracellular Ca2+ properties were assessed as peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR90), maximal velocity of shortening/relengthening (+/-dL/dt), and intracellular Ca2+ decay (tau). HG myocytes exhibited normal PS; decreased +/-dL/dt; and prolonged TPS, TR90, and tau. Interestingly, the HG-induced abnormalities were prevented with the AT1 blocker L-158,809 (10 to 1000 nmol/L) but not the Janus kinase-2 (JAK2) inhibitor AG-490 (10 to 100 micromol/L). The only effect of AT1 blockade on NG myocytes was enhanced PS at 1000 nmol/L. AT1 antagonist-elicited cardiac protection against HG was nullified by the NADPH oxidase activator sodium dodecyl sulfate (80 micromol/L) and mimicked by the NADPH oxidase inhibitors diphenyleneiodonium (10 micromol/L) or apocynin (100 micromol/L). Western blot analysis confirmed that the protein abundance of NADPH oxidase subunit p47phox and the AT1 but not the AT2 receptor was enhanced in HG myocytes. In addition, the HG-induced increase of p47phox was prevented by L-158,809. Enhanced reactive oxygen species production observed in HG myocytes was prevented by AT1 blockade or NADPH oxidase inhibition. Collectively, our data suggest that local Ang II, acting via AT1 receptor-mediated NADPH oxidase activation, is involved in hyperglycemia-induced cardiomyocyte dysfunction, which might play a role in diabetic cardiomyopathy.     

Demonstration of altered fibroblast contractile activity in hypertensive heart disease

OBJECTIVE: The aim of this study is to investigate the idea that altered fibroblast contractile activity is involved in the pathogenesis of hypertensive heart disease (HHD). METHODS: Cell area and contraction are quantified using the traction force microscopy technique for cardiac fibroblasts isolated from both normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. RESULTS: The data indicate that there are marked phenotypic differences between the two cell types. For instance, WKY fibroblasts exert an average traction stress of approximately 3.3 kPa and have an area of approximately 2640 microm(2). Under identical conditions the SHR fibroblasts have an area approximately 1.45 times larger (p<0.01) and exert an average stress approximately 1.86 times higher (p<0.01). Challenging WKY fibroblasts with 1 micromol/l angiotensin II (Ang II) gradually causes a approximately 2-fold increase in traction after 1 h while simultaneously causing a approximately 28% decrease in area. In contrast, Ang II has no effect on SHR fibroblasts. The data also show that WKY and SHR cells respond in different ways when challenged with irbesartan (Irb). The addition of 1 micromol/l Irb initially causes WKY cells to decrease their average traction output by approximately 50% after approximately 10 min. Subsequently, contractile activity begins to recover and returns to normal after 1 h. The SHR cells also decrease their tractions by approximately 50%, but this decrease requires 30 min for completion and there is no recovery to the initial contractile state. For both cell types, Irb produces no significant effect on area and the combined effect of equimolar Irb and Ang II is the same as Irb alone. CONCLUSION: These in vitro data suggest that among the many factors producing hypertensive heart disease in SHR's are excessive contraction of their cardiac fibroblasts and defective control of fibroblast contraction by Ang II.     

Appraisal of the role of angiotensin II and aldosterone in ventricular myocyte apoptosis in adult normotensive rat

Despite previous observations on isolated ventricular myocytes, there are still few evidences that angiotensin II induces cardiomyocyte apoptosis in vivo. The possibility that aldosterone, the final hormone of the renin-angiotensin-aldosterone system under Ang II control, can stimulate cardiac apoptosis has not yet been explored. Angiotensin II or aldosterone (1mg/kg each) were infused in adult normotensive rats for different times, and the number of apoptotic ventricular myocyte nuclei was quantified by the TUNEL method, along with caspase-3 activation. The role of angiotensin II type 1 receptor in vivo was assessed by selective blockade with valsartan and ex vivo by binding experiments. In addition, myocytes in primary culture were incubated with Ang II or aldosterone in presence of spironolactone. Continuous infusion of Ang II induced a rapid, AT(1)-mediated increase of apoptotic cardiomyocyte nuclei (from 14+/-9 to 188+/-35 TdT-labeled nuclei/10(6) after 3h, P<0.005) and of activated caspase-3, that normalized after 24h. The normalization was associated with a down-regulation of myocardial AT(1) receptors. Aldosterone stimulated cardiomyocyte apoptosis both in vivo and in isolated cells, to a similar extent as Ang II. The maximal apoptotic rate reported here ( approximately 0.02%) and the transient effect of Ang II suggest that myocyte loss by apoptosis is limited in the present model. The data on aldosterone-induced ventricular myocyte apoptosis deserve further attention to delineate the role of aldosterone in cell death and offer possible mechanistic explanations on the benefits afforded by aldosterone receptor antagonists in heart failure.     

Mildly oxidized low-density lipoprotein acts synergistically with angiotensin II in inducing vascular smooth muscle cell proliferation

OBJECTIVES: Considerable attention has been focused on both mildly oxidized low-density lipoprotein (mox-LDL) and highly oxidized LDL (ox-LDL) as important risk factors for cardiovascular disease. Further, angiotensin II (Ang II) appears to play a crucial role in the development of hypertension and atherosclerosis. We assessed the effect of oxidatively modified LDL and its major oxidative components, i.e., hydrogen peroxide (H2O2), lysophosphatidylcholine (LPC), and 4-hydroxy-2-nonenal (HNE) and their interaction with Ang II on vascular smooth muscle cell (VSMC) DNA synthesis. METHODS: Growth-arrested rabbit VSMCs were incubated in serum-free medium with different concentrations of native LDL, mox-LDL, ox-LDL, H2O2, LPC, or HNE with or without Ang II. DNA synthesis in VSMCs was measured by [3H]thymidine incorporation. RESULTS: Ang II stimulated DNA synthesis in a dose-dependent manner with a maximal effect at a concentration of 1 micromol/l (173%). Ang II (0.5 micromol/l) amplified the effect of native LDL at 500 ng/ml, ox-LDL at 100 ng/ml, and mox-LDL at 50 ng/ml on DNA synthesis (108 to 234%, 124 to 399%, 129 to 433%, respectively). H2O2 had a maximal effect at a concentration of 5 micromol/l (177%), LPC at 15 micromol/l (156%), and HNE at 0.5 micromol/l (137%). Low concentrations of H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) also acted synergisitically with Ang II (0.5 micromol/l) in inducing DNA synthesis to 308, 304, or 238%, respectively. Synergistic interactions of Ang II (0.5 micromol/l) with mox-LDL, ox-LDL (both 50 ng/ml), H2O2 (1 micromol/l), LPC (5 micromol/l), or HNE (0.1 micromol/l) on DNA synthesis were completely reversed by the combined use of probucol (10 micromol/l), a potent antioxidant and candesartan (0.1 micromol/l), an AT1 receptor antagonist. CONCLUSIONS: Our results suggest that mox-LDL, ox-LDL, and their major components H2O2, LPC, and HNE act synergistically with Ang II in inducing VSMC DNA synthesis. A combination of antioxidants with AT1 receptor blockade may be effective in the treatment of VSMC proliferative disorders associated with hypertension and atherosclerosis.     

Effects of angiotensin-(1-7) and other bioactive components of the renin-angiotensin system on vascular resistance and noradrenaline release in rat kidney

OBJECTIVE: Angiotensin (Ang) is broken down enzymatically to several different metabolites which, in addition to Ang II, may have important biological effects in the kidney. This study investigates the role of Ang metabolites on vascular resistance and noradrenaline release in the rat kidney. METHODS AND RESULTS: In rat isolated kidney Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I induced pressor responses and enhanced noradrenaline release to renal nerve stimulation (RNS) in an concentration-dependent manner, with the following rank order of potency (EC(50)): Ang II >or= Ang III > Ang I = des-Asp-Ang I > Ang IV. All effects were blocked by the AT(1)-receptor antagonist EXP 3174 (0.1 micromol/l) but not by the AT(2)-receptor antagonist PD 123319 (1 micromol/l). Angiotensin-converting enzyme (ACE) inhibition by captopril (10 micromol/l) abolished the effect of Ang I and des-Asp-Ang I but had no influence on the effect of the other metabolites. Ang-(1-7) blocked the effects of Ang I and Ang II, being 10 times more potent against Ang I than Ang II. The selective Ang-(1-7) receptor blocker d-Ala7-Ang-(1-7) (10 micromol/l) did not influence the inhibitory effects of Ang-(1-7). Ang-(1-7) (10 micromol/l) by itself had no influence on vascular resistance and RNS-induced noradrenaline release. CONCLUSION: Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I regulate renal vascular resistance and noradrenaline release by activation of AT(1) receptors. In the case of Ang I and des-Asp-Ang I this depends on conversion by ACE. Ang-(1-7) may act as a potent endogenous inhibitor/antagonist of ACE and the AT(1)-receptors, respectively.