Characterization of the PGI2/IP system in cultured rat mesangial cells

Mesangial cells play an important role in glomerular function. They are an important source of cyclooxygenase (COX)-derived arachidonic acid metabolites, including prostaglandin E(2) and prostacyclin. Prostacyclin receptor (IP) mRNA was amplified from cultured mesangial cell total RNA by RT-PCR. While the prostaglandin E(2) receptor subtype EP(2) was not detected, EP(1,3,4) mRNA was amplified. Also, IP protein was noted in mesangial cells, proximal tubules, inner medullary collecting ducts, and the inner and outer medulla. But no protein was detected in whole cortex preparations. Prostacyclin analogues: cicaprost and iloprost, increased cAMP levels in mesangial cells. On the other hand, arginine-vasopressin and angiotensin II increased intracellular calcium in mesangial cells, but cicaprost, iloprost and prostaglandin E(2) had no effect. Moreover, a 50% inhibition of cicaprost- and iloprost-cAMP stimulation was observed upon mesangial cell exposure to 25 and 35 mM glucose for 5 days. But no change in IP mRNA was observed at any glucose concentration or time exposure. Although 25 mM glucose had no effect on COX-1 protein levels, COX-2 was increased up to 50%. In contrast, PGIS levels were reduced by 50%. Thus, we conclude that the prostacyclin/IP system is present in cultured rat mesangial cells, coupling to a cAMP stimulatory pathway. High glucose altered both enzymes in the PGI(2) synthesis pathway, increasing COX-2 but reducing PGIS. In addition, glucose diminished the cAMP response to prostacyclin analogues. Therefore, glucose attenuates the PGI(2)/IP system in cultured rat mesangial cells.     

活性氧介导内皮素-1诱导的培养新生大鼠心肌细胞肥大

实验在原代培养的新生大鼠心肌细胞中进行,检测内皮素-1(endothelin-1,ET-1)及其他药物对心肌细胞活性氧(reactiveoxygen species,ROS)产生和心肌细胞肥大的作用,以探讨ROS在ET-1诱导的心肌细胞肥大信号通路中的作用及ROS与蛋白激酶C(protein kinase C,PKC)活化的关系。细胞内ROS水平用ROS敏感的荧光探针2,7-dichlorofluorescin dictate(DCF-DA)反映,心肌细胞肥大通过细胞内RNA含量、细胞内蛋白质含量、细胞表面积大小来确定。实验结果如下:单独使用ET-1后,心肌细胞内反应ROS含量的DCF-DA荧光值比对照组增加77%,反应心肌肥大的PI荧光值、细胞内蛋白质含量、细胞表面积也分别比对照组增加128%、87%和151%。ET-1合用内皮素受体A亚型(ET_A)受体拮抗剂ABT-627、PKC抑制剂CC或过氧化氢酶后,DCF-DA的增加分别减弱62%、60%和51%,同时心肌细胞肥大也被抑制,单独使用PKC激动剂佛波醇脂(PMA)也能使DCF-DA的产生比对照组增加74%。因此,在ET-1诱导心肌细胞肥大的过程中,ET-1能够使心肌细胞产生ROS和诱导ROS依赖的心肌细胞肥大,这一作用依赖于ET_A受体的激活和PKC的活化,·ROS在ET-1诱导心肌细胞肥大中起信号传递的作用。     

Endothelin-1 attenuates increases in hydraulic conductivity due to platelet-activating factor via prostacyclin release

We previously showed that endothelin-1 (ET-1) and prostacyclin (PGI(2)) similarly attenuate increases in microvascular permeability induced by platelet-activating factor (PAF). This led us to hypothesize that ET-1 attenuates trans-endothelial fluid flux during PAF through PGI(2) release. We tested this hypothesis in three phases. First, bovine pulmonary artery endothelial cells were exposed to 0.008-8 μM ET-1 and assayed for PGI(2) release. Second, to determine whether increased transmonolayer flux after PAF could be attenuated by ET-1 or PGI(2) and reversed by PGI(2) synthesis inhibition or PGI(2) receptor blockade, we measured endothelial cell transmonolayer flux after cells were exposed to 10 nM PAF plus 10 μM PGI(2) or 80 pM ET-1, with or without 500 μM tranylcypromine (PGI(2) synthase inhibitor) or 20 μM CAY-10441 (PGI(2) receptor blocker). Finally, hydraulic conductivity (L(p)) was measured in rat mesenteric venules in vivo after exposure to 10 nM PAF and 80 pM ET-1 with or without tranylcypromine (100 and 500 μM) or CAY-10441 (2 and 20 μM). We found that in vitro, ET-1 stimulated a dose-dependent increase in PGI(2) production (from 126 to 217 pg/ml, P < 0.01). Compared with PAF alone, PGI(2) plus PAF and ET-1 plus PAF decreased transmonolayer flux similarly by 52 and 46%, respectively (P < 0.01), while tranylcypromine and CAY-10441 reversed these effects by 92 and 47%, respectively (P < 0.05). In vivo, PAF increased L(p) fourfold (P < 0.01) and ET-1 attenuated this effect by 83% (P < 0.01). Tranylcypromine and CAY-10441 reversed the ET-1 attenuation in L(p) during PAF by 55 and 45%, respectively (P < 0.01). We conclude that ET-1 may stimulate endothelial cell PGI(2) release to attenuate the increases in transmonolayer flux and hydraulic conductivity secondary to PAF.     

Investigation of the agonist activity of prostacyclin analogues on prostanoid EP4 receptors using GW 627368 and taprostene: evidence for species differences

The possibility that the prostacyclin analogues AFP-07 and cicaprost relax saphenous vein preparations of pig, guinea-pig and rabbit by simultaneously activating prostanoid EP4 and IP (prostacyclin) receptors was investigated using the high-affinity EP4 antagonist GW 627368. The IP receptor system in each preparation was suppressed with the partial agonist taprostene. The results indicate that AFP-07 and cicaprost are moderately potent EP4 agonists on pig saphenous vein, but much weaker EP4 agonists on guinea-pig saphenous vein. GW 627368 did not antagonise AFP-07- and cicaprost-induced relaxation of rabbit saphenous vein, which contrasts with a previous study using the EP4 blocker AH 23848. However, treatment with taprostene was of less value due to poorer antagonism of the rabbit IP system; this may be related to the presence of a sensitive EP2 relaxant system. Relaxation of each preparation induced by the selective EP2 agonist ONO-AE1-259 was unaffected by GW 627368, with and without taprostene.     

Involvement of peptidyl-prolyl isomerase Pin1 in the inhibitory effect of fluvastatin on endothelin-1-induced cardiomyocyte hypertrophy

Aims

Cardiac hypertrophy is elicited by endothelin (ET)-1 as well as other neurohumoral factors, hemodynamic overload, and oxidative stress; HMG-CoA reductase inhibitors (statins) were shown to inhibit cardiac hypertrophy partly via the anti-oxidative stress. One of their common intracellular pathways is the phosphorylation cascade of MEK signaling. Pin1 specifically isomerizes the phosphorylated protein with Ser/Thr-Pro bonds and regulates their activity through conformational changes. There is no report whether the Pin1 activation contributes to ET-1-induced cardiomyocyte hypertrophy and whether the Pin1 inactivation contributes to the inhibitory effect of statins. The aim of this study was to reveal these questions.

Main methods

We assessed neonatal rat cardiomyocyte hypertrophy using ET-1 and fluvastatin by the cell surface area, ANP mRNA expression, JNK and c-Jun phosphorylation, and [³H]-leucine incorporation.

Key findings

Fluvastatin inhibited ET-1-induced increase in the cell surface area, ANP expression, and [³H]-leucine incorporation; and it suppressed the signaling cascade from JNK to c-Jun. The phosphorylated Pin1 level, an inactive form, was decreased by ET-1; however, it reached basal level by fluvastatin. Furthermore, Pin1 overexpression clearly elicited cardiomyocyte hypertrophy, which was inhibited by fluvastatin.

Significance

This is the first report that ET-1-induced cardiomyocyte hypertrophy is mediated through the Pin1 activation and that the inhibitory effect of fluvastatin on cardiomyocyte hypertrophy would partly be attributed to the suppression of the Pin1 function. This study firstly suggests that Pin1 determines the size of hypertrophied cardiomyocyte by regulating the activity of phosphorylated molecules and that statins exert their pleiotropic effects partly via Pin1 inactivation.     

Contributory role of VEGF overexpression in endothelin-1-induced cardiomyocyte hypertrophy

Although endothelin-1 (ET-1) stimulates vascular endothelial growth factor (VEGF) expression in a variety of cells, including endothelial cells and vascular smooth muscle cells, the effects of ET-1 on expression of VEGF and its receptors in cardiomyocytes are unknown. In the present study, we found that treatment of neonatal rat cardiomyocytes with ET-1 for 24 h resulted in upregulation of VEGF and its two principal receptors, fetal liver kinase 1 and fms-like tyrosine kinase 1, in a concentration-dependent manner (10(-12) to 10(-6) M). ET-1 treatment also caused significant cardiomyocyte hypertrophy, as indicated by increases in cell surface area and [(14)C]leucine uptake by cardiomyocytes. Treatment with TA-0201 (10(-6) M), an ET(A)-selective blocker, eliminated ET-1-induced overexpression of VEGF and its receptors as well as cardiomyocyte hypertrophy. Treatment with VEGF neutralizing peptides (5-10 mug/ml) partially but significantly inhibited ET-1-induced cardiomyocyte hypertrophy. These results suggest that ET-1 treatment of cardiomyocytes promotes overexpression of VEGF and its receptors via activation of ET(A) receptors, and consequently the upregulated VEGF signaling system appears to contribute, at least in part, to ET-1-induced cardiomyocyte hypertrophy.     

Eicosapentaenoic acid prevents endothelin-1-induced cardiomyocyte hypertrophy in vitro through the suppression of TGF-beta 1 and phosphorylated JNK

The cardiovascular benefit of fish oil in humans and experimental animals has been reported. Endothelin (ET)-1 is a well-known cardiac hypertrophic factor. However, although many studies link a fish oil extract, eicosapentaenoic acid (EPA), to cardiac protection, the effects of EPA on cardiac hypertrophy and underlying mechanism(s) are unclear. The present study investigated whether EPA prevents ET-1-induced cardiomyocyte hypertrophy; the potential pathways likely to underlie such an effect were also investigated. Cardiomyocytes were isolated from neonatal rat heart, cultured for 3 days, and then treated for 24 h with vehicle only (control), treated with 0.1 nM ET-1 only, or pretreated with 10 microM EPA and then treated with 0.1 nM ET-1. The cells were harvested, and changes in cell surface area, protein synthesis, expression of a cytoskeletal (alpha-actinin) protein, and cell signaling were analyzed. ET-1 induced a 97% increase in cardiomyocyte surface area, a 72% increase in protein synthesis rate, and an increase in expression of alpha-actinin and signaling molecule [transforming growth factor-beta 1 (TGF-beta 1), c-Jun NH2-terminal kinase (JNK), and c-Jun]. Development of these ET-1-induced cellular changes was attenuated by EPA. Moreover, the hypertrophied cardiomyocytes showed a 1.5- and a 1.7-fold increase in mRNA expression of atrial and brain natriuretic peptides, the classical molecular markers of cardiac hypertrophy, respectively; these changes were also suppressed by EPA. Here we show that ET-1 induces cardiomyocyte hypertrophy and expression of hypertrophic markers, possibly mediated by JNK and TGF-beta 1 signaling pathways. These ET-1-induced effects were blocked by EPA, a major fish oil ingredient, suggesting that fish oil may have beneficial protective effects on cardiac hypertrophy.     

Dimerization of the human receptors for prostacyclin and thromboxane facilitates thromboxane receptor-mediated cAMP generation

Prostacyclin (PGI(2)) and thromboxane (TxA(2)) are biological opposites; PGI(2), a vasodilator and inhibitor of platelet aggregation, limits the deleterious actions of TxA(2), a vasoconstrictor and platelet activator. The molecular mechanisms involved in the counterregulation of PGI(2)/TxA(2) signaling are unclear. We examined the interaction of the receptors for PGI(2) (IP) and TxA(2) (TPalpha). IP-induced cAMP and TP-induced inositol phosphate generation were unaltered when the receptors were co-expressed in HEK 293 cells (IP/TPalpha-HEK). TP-cAMP generation, in response to TP agonists or a TP-dependent isoprostane, iPE(2)III, was evident in IP/TPalpha-HEK and in aortic smooth muscle cells, but not in cells expressing either receptor alone, or in IP-deficient aortic smooth muscle cells. Augmentation of TP-induced cAMP generation, with the IP agonist cicaprost, was ablated in IP-deficient cells and was independent of direct IP signaling. IP/TPalpha heterodimers were formed constitutively when the receptors were co-expressed, with no overt changes in ligand binding to the individual receptor sites. However, despite inefficient binding of iPE(2)III to either the IP or TPalpha, expressed alone or in combination, robust cAMP generation was evident in IP/TPalpha-HEK, suggesting the formation of an alternative receptor site. Thus, IP/TPalpha dimerization was coincident with TP-cAMP generation, promoting a "PGI(2)-like" cellular response to TP activation. This represents a previously unknown mechanism by which IP may limit the cellular effects of TP.     

Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells

Signaling through the PGI(2) receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI(2.) In this study, we determined that PGI(2) analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI(2) analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI(2) analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-alpha, IL-1alpha, IL-6) and chemokines (MIP-1alpha, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-kappaB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI(2) signaling through the IP may exert anti-inflammatory effects by acting on DC.     

Effect of pravastatin on development of left ventricular hypertrophy in spontaneously hypertensive rats

Endothelin (ET)-1 has been implicated in the development of cardiac hypertrophy. We investigated the effect of pravastatin on development of ventricular hypertrophy in spontaneously hypertensive rats (SHR) and whether the attenuated hypertrophic effect was via reduced ET-1 expression. Normolipidemic SHR were treated with one of the following therapies for 8 wk: vehicle, the nonselective ET receptor antagonists bosentan, pravastatin, mevalonate, hydralazine, or combination of pravastatin + mevalonate from the age of 8 wk at the very early stage of cardiac hypertrophy. Treatment with bosentan and pravastatin significantly decreased left ventricular mass index for body weight and cardiomyocyte sizes isolated by enzymatic dissociation. The myocardial ET-1 levels and preproET-1 mRNA assessed using real-time quantitative RT-PCR were significantly higher (both P < 0.001) in the SHR compared with Wistar-Kyoto rats. The increased tissue ET-1 levels can be inhibited after pravastatin administration. Immunohistochemical analysis confirmed the changes of ET-1. Left ventricular mass index for body weight correlated positively with tissue ET-1 levels (P = 0.0004). A dissociation between the effects of blood pressure and cardiac structure was noted, because pravastatin and hydralazine reduced arterial pressure similarly. Pravastatin-induced effects were reversed by the addition of mevalonate. In conclusion, these results suggest a crucial role of cardiac endothelin system in the early development of ventricular hypertrophy in the SHR. Pravastatin is endowed with cardiac antihypertropic properties that are independent of its hemodynamic and hypolipidemic effects and appear to be related to their capacity to decrease cardiac ET-1 levels, which is linked to mevalonate metabolism.     

Prostacyclin attenuates oxidative damage of myocytes by opening mitochondrial ATP-sensitive K+ channels via the EP3 receptor

Prostacyclin (PGI2) and the PGE family alleviate myocardial ischemia-reperfusion injury and limit oxidative damage. The cardioprotective effects of PGI2 have been traditionally ascribed to activation of IP receptors. Recent advances in prostanoid research have revealed that PGI2 can bind not only to IP, but also to EP, receptors, suggesting cross talk between PGI2 and PGEs. The mechanism(s) whereby PGI2 protects myocytes from oxidative damage and the specific receptors involved remain unknown. Thus fresh isolated adult rat myocytes were exposed to 200 microM H2O2 with or without carbaprostacyclin (cPGI2), IP-selective agonists, and ONO-AE-248 (an EP3-selective agonist). Cell viability was assessed by trypan blue exclusion after 30 min of H2O2 superfusion. cPGI2 and ONO-AE-248 significantly improved cell survival during H2O2 superfusion; IP-selective agonists did not. The protective effect of cPGI2 and ONO-AE-248 was completely abrogated by pretreatment with 5-hydroxydecanoate or glibenclamide. In the second series of experiments, the mitochondrial ATP-sensitive K+ (K(ATP)) channel opener diazoxide (Dx) reversibly oxidized flavoproteins in control myocytes. Exposure to prostanoid analogs alone had no effect on flavoprotein fluorescence. A second application of Dx in the presence of cPGI2 or ONO-AE-248 significantly increased flavoprotein fluorescence compared with Dx alone, but IP-selective agonists did not. This study demonstrates that PGI2 analogs protect cardiac myocytes from oxidative stress mainly via activation of EP3. The data also indicate that activation of EP3 receptors primes the opening of mitochondrial K(ATP) channels and that this mechanism is essential for EP3-dependent protection.     

The role of angiotensin II,endothelin-1 and transforming growth factor-β as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy

Cardiac hypertrophy is a compensatory response of myocardial tissue upon increased mechanical load. Of the mechanical factors, stretch is rapidly followed by hypertrophic responses. We tried to elucidate the role of angiotensin II (AII), endothelin-1 (ET-1) and transforming growth factor- (TGF-) as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy. We collected conditioned medium (CM) from stretched cardiomyocytes and from other stretched cardiac cells, such as cardiac fibroblasts, endothelial cells and vascular smooth muscle cells (VSMCs). These CMs were administered to stationary cardiomyocytes with or without an AII type 1 (AT₁) receptor antagonist (losartan), an ET-1 type A (ET_A) receptor antagonist (BQ610), or anti-TGF- antibodies. By measuring the mRNA levels of the proto-oncogene c-fos and the hypertrophy marker gene atrial natriuretic peptide (ANP), the molecular phenotype of the CM-treated stationary cardiomyocytes was characterized.Our results showed that c-fos and ANP expression in stationary cardiomyocytes was increased by AII release from cardiomyocytes that had been stretched for 60 min. Stretched cardiomyocytes, cardiac fibroblasts and endothelial cells released ET-1 which led to increased c-fos and ANP expression in stationary cardiomyocytes. ET-1 released by stretched VSMCs, and TGF- released by stretched cardiac fibroblasts and endothelial cells, appeared to be paracrine mediators of ANP expression in stationary cardiomyocytes.These results indicate that AII, ET-1 and TGF- (released by cardiac and vascular cell types) act as autocrine/paracrine mediators of stretch-induced cardiomyocyte hypertrophy. Therefore, it is likely that in stretched myocardium the cardiomyocytes, cardiac fibroblasts, endothelial cells and VSMCs take part in intercellular interactions contributing to cardiomyocyte hypertrophy.     

G蛋白,蛋白激酶C和Na^＋—H^＋交换在内皮素—1诱导培养心肌细胞肥大反应中的

内皮系-1（ET-1）是一种强的生长因子,并诱导心肌细胞肥大反应。在本实验中,我们探讨了G蛋白、蛋白激酶C（PKC）和Na＋－H＋交换在ET-1诱导的培养新生大鼠心肌细胞肥大反应中的作用。ET-1（10－10～10－7mol／L）促进3H－亮氨酸掺入,增加细胞蛋白质的含量和心肌细胞的表面积,且呈剂量依赖性,它们的EC50分别为5．2×10-10,5．2×10－10和7．3×10－10mol／L。用蛋白激酶C（PKC）抑制剂,Staurosporin（2nmol／L）预处理心肌细胞,可完全阻断ET-1诱导的心肌细胞的这些肥大反应,而蛋白激酶C激动剂,佛波酸酯（PMA）（10－8～10－6mol／L）呈剂量依赖性促进心肌细胞的肥大反应。用Na＋－H＋交换抑制剂,氨氯毗咪（10-4mol／L）预处理心肌细胞,可抑制ET－1诱导的心肌细胞肥大反应,但不影响PMA诱导的心肌细胞肥大反应。百日咳毒素（150ng／ml）预处理心肌细胞,可明显抑制ET－1诱导的心肌细胞肥大反应。这些结果提示,ET-1诱导的培养新生大鼠心肌细胞肥大反应是与百日咳毒素敏感的G蛋白相耦联,蛋白激酶C和Na＋．H＋交换可能在ET-1诱导的心肌细胞肥大反应中是重要的细胞内信使转导途径。     

Effect of eicosapentaenoic acid on the different endothelin system components in endothelin-1-induced hypertrophied cardiomyocytes

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro-ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1-induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1-administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco's modified Eagle's medium and Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1-treated (0.1 nM) group, and ET-1-treated group pretreated with EPA (10 microM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro-ET-1 mRNA expression was 53% upregulated in ET-1-induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1-administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET(A) and ET(B), tended to be increased in the ET-1-treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro-ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1-induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.     

Nitroxyl (HNO) stimulates soluble guanylyl cyclase to suppress cardiomyocyte hypertrophy and superoxide generation

Background

New therapeutic targets for cardiac hypertrophy, an independent risk factor for heart failure and death, are essential. HNO is a novel redox sibling of NO• attracting considerable attention for the treatment of cardiovascular disorders, eliciting cGMP-dependent vasodilatation yet cGMP-independent positive inotropy. The impact of HNO on cardiac hypertrophy (which is negatively regulated by cGMP) however has not been investigated.

Methods

Neonatal rat cardiomyocytes were incubated with angiotensin II (Ang II) in the presence and absence of the HNO donor Angeli''s salt (sodium trioxodinitrate) or B-type natriuretic peptide, BNP (all 1 µmol/L). Hypertrophic responses and its triggers, as well as cGMP signaling, were determined.

Results

We now demonstrate that Angeli''s salt inhibits Ang II-induced hypertrophic responses in cardiomyocytes, including increases in cardiomyocyte size, de novo protein synthesis and β-myosin heavy chain expression. Angeli''s salt also suppresses Ang II induction of key triggers of the cardiomyocyte hypertrophic response, including NADPH oxidase (on both Nox2 expression and superoxide generation), as well as p38 mitogen-activated protein kinase (p38MAPK). The antihypertrophic, superoxide-suppressing and cGMP-elevating effects of Angeli''s salt were mimicked by BNP. We also demonstrate that the effects of Angeli''s salt are specifically mediated by HNO (with no role for NO• or nitrite), with subsequent activation of cardiomyocyte soluble guanylyl cyclase (sGC) and cGMP signaling (on both cGMP-dependent protein kinase, cGK-I and phosphorylation of vasodilator-stimulated phosphoprotein, VASP).

Conclusions

Our results demonstrate that HNO prevents cardiomyocyte hypertrophy, and that cGMP-dependent NADPH oxidase suppression contributes to these antihypertrophic actions. HNO donors may thus represent innovative pharmacotherapy for cardiac hypertrophy.     

Vasopressin accelerates protein synthesis in neonatal rat cardiomyocytes

Arginine vasopressin (AVP) has been shown to promote vascular smooth muscle cell hypertrophy and hyperplasia of fibroblasts. The present study examines the effect of AVP and endothelin-1 (ET-1) on protein, DNA, and RNA synthesis in primary cultures of serum deprived neonatal rat cardiomyocytes (RC) as assessed by changes in [3H] phenylalanine, [3H] thymidine, and [14C] uridine incorporation respectively. Both AVP and ET-1 evoked significant increases in protein synthesis in RC of 36 ± 12% (p < 0.05) and 53 ± 22% (p < 0.01) respectively. The stimulating action of AVP on [3H] phenylalanine incorporation was abolished by pretreatment with 2-nitro-4carboxyphenyl-N, N-diphenylcarbamate (NCDC), a phospholipase C (PLC) inhibitor. [14C] uridine incorporation was significantly higher in cells incubated with ET-1 (95 ± 12%) but not AVP (9 ± 11%). Neither AVP nor ET-1 significantly affected cell number or [3H] thymidine incorporation, suggesting a lack of a hyperplastic effect. AVP evoked an increase in [Ca2+]i levels (162 ± 12 nmol/L from a basal value of 77 ± 6 nmol/L) which was completely abolished by pretreatment with either NCDC or cyclopiazonic acid (sarcoplasmic reticulum (SR) Ca2+ pump inhibitor) but unaffected by ryanodine (ryanodine sensitive SR Ca2+ store depletor). Taken together, these data suggest that AVP, in a PLC dependent manner, stimulates both protein synthesis and augments [Ca2+]i release in RC from ryanodine insensitive (IP3 sensitive) Ca2+ stores. Thus, AVP may promote cardiac hypertrophy via direct effects on cardiomyocyte protein synthesis secondary to IP3 mediated [Ca2+]i release.     

Myocardial dysfunction and neurohumoral activation without remodeling in left ventricle of monocrotaline-induced pulmonary hypertensive rats

In monocrotaline (MCT)-induced pulmonary hypertension (PH), only the right ventricle (RV) endures overload, but both ventricles are exposed to enhanced neuroendocrine stimulation. To assess whether in long-standing PH the left ventricular (LV) myocardium molecular/contractile phenotype can be disturbed, we evaluated myocardial function, histology, and gene expression of autocrine/paracrine systems in rats with severe PH 6 wk after subcutaneous injection of 60 mg/kg MCT. The overloaded RV underwent myocardial hypertrophy (P < 0.001) and fibrosis (P = 0.014) as well as increased expression of angiotensin-converting enzyme (ACE) (8-fold; P < 0.001), endothelin-1 (ET-1) (6-fold; P < 0.001), and type B natriuretic peptide (BNP) (15-fold; P < 0.001). Despite the similar upregulation of ET-1 (8-fold; P < 0.001) and overexpression of ACE (4-fold; P < 0.001) without BNP elevation, the nonoverloaded LV myocardium was neither hypertrophic nor fibrotic. LV indexes of contractility (P < 0.001) and relaxation (P = 0.03) were abnormal, however, and LV muscle strips from MCT-treated compared with sham rats presented negative (P = 0.003) force-frequency relationships (FFR). Despite higher ET-1 production, BQ-123 (ET(A) antagonist) did not alter LV MCT-treated muscle strip contractility distinctly (P = 0.005) from the negative inotropic effect exerted on shams. Chronic daily therapy with 250 mg/kg bosentan (dual endothelin receptor antagonist) after MCT injection not only attenuated RV hypertrophy and local neuroendocrine activation but also completely reverted FFR of LV muscle strips to positive values. In conclusion, the LV myocardium is altered in advanced MCT-induced PH, undergoing neuroendocrine activation and contractile dysfunction in the absence of hypertrophy or fibrosis. Neuroendocrine mediators, particularly ET-1, may participate in this functional deterioration.     

Role of endothelin in the induction of cardiac hypertrophy in vitro

Endothelin (ET-1) is a peptide hormone mediating a wide variety of biological processes and is associated with development of cardiac dysfunction. Generally, ET-1 is regarded as a molecular marker released only in correlation with the observation of a hypertrophic response or in conjunction with other hypertrophic stress. Although the cardiac hypertrophic effect of ET-1 is demonstrated, inotropic properties of cardiac muscle during chronic ET-1-induced hypertrophy remain largely unclear. Through the use of a novel in vitro multicellular culture system, changes in contractile force and kinetics of rabbit cardiac trabeculae in response to 1 nM ET-1 for 24 hours can be observed. Compared to the initial force at t = 0 hours, ET-1 treated muscles showed a ∼2.5 fold increase in developed force after 24 hours without any effect on time to peak contraction or time to 90% relaxation. ET-1 increased muscle diameter by 12.5±3.2% from the initial size, due to increased cell width compared to non-ET-1 treated muscles. Using specific signaling antagonists, inhibition of NCX, CaMKII, MAPKK, and IP3 could attenuate the effect of ET-1 on increased developed force. However, among these inhibitions only IP3 receptor blocker could not prevent the increase muscle size by ET-1. Interestingly, though calcineurin-NFAT inhibition could not suppress the effect of ET-1 on force development, it did prevent muscle hypertrophy. These findings suggest that ET-1 provokes both inotropic and hypertrophic activations on myocardium in which both activations share the same signaling pathway through MAPK and CaMKII in associated with NCX activity.     

Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart

The serine-threonine kinase, Akt, inhibits cardiomyocyte apoptosis acutely both in vitro and in vivo. However, the effects of chronic Akt activation in the heart are unknown. To address this issue, we generated transgenic mice (TG+) with cardiac-specific expression of a constitutively active mutant of Akt (myr-Akt) driven by the myosin heavy chain-alpha promoter. Three TG+ founders (9-19 weeks) died suddenly with massive cardiac dilatation. Two viable TG+ lines (TG564 and TG20) derived from independent founders demonstrated cardiac-specific transgene expression as well as activation of Akt and p70S6 kinase. TG564 (n = 19) showed cardiac hypertrophy with a heart/body weight ratio 2.3-fold greater than littermates (n = 17, p < 0.005). TG20 (n = 18) had less marked cardiac hypertrophy with a heart/body weight ratio 1.6-fold greater than littermates (n = 17, p < 0.005). Isolated TG564 myocytes were also hypertrophic with surface areas 1.7-fold greater than littermates (p < 0.000001). Echocardiograms in both lines demonstrated concentric hypertrophy and preserved systolic function. After ischemia-reperfusion, TG+ had a 50% reduction in infarct size versus TG- (17 +/- 3% versus 34 +/- 4%, p < 0.001). Thus, chronic Akt activation is sufficient to cause a spectrum of phenotypes from moderate cardiac hypertrophy with preserved systolic function and cardioprotection to massive cardiac dilatation and sudden death.