Activation of peroxisome proliferator-activated receptor gamma inhibits endothelin-1-induced cardiac hypertrophy via the calcineurin/NFAT signaling pathway

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) has been described as a negative regulator of cardiac hypertrophy. A better understanding of PPAR-gamma and cardiac hypertrophy may facilitate the development of novel therapeutic strategies to treat heart diseases related to cardiac hypertrophy by mimicking the naturally preferred mechanisms. In the present study, we investigated the interaction between PPAR-gamma and calcineurin/nuclear factor of activated T-cells (NFAT) in endothelin-1 (ET-1)-induced hypertrophy of neonatal rat cardiac myocytes. The results suggest that the treatment of cultured cardiac myocytes with a PPAR-gamma ligand, rosiglitazone, inhibited the ET-1-induced increase in protein synthesis, surface area, calcineurin enzymatic activity, and protein expression. Both the application of rosiglitazone and overexpression of the PPAR-gamma inhibited the nuclear translocation of NFATc4. Moreover, co-immunoprecipitation studies showed that rosiglitazone enhanced the association between PPAR-gamma and calcineurin/NFAT. These results suggest that ET-1-induced cardiac hypertrophy is inhibited by activation of PPAR-gamma, which is at least partly due to cross-talk between PPAR-gamma and calcineurin/NFAT.     

Localization of functional endothelin receptor signaling complexes in cardiac transverse tubules

Endothelin-1 (ET-1) is an autocrine factor in the mammalian heart important in enhancing cardiac performance, protecting against myocardial ischemia, and initiating the development of cardiac hypertrophy. The ETA receptor is a seven-transmembrane G-protein-coupled receptor whose precise subcellular localization in cardiac muscle is unknown. Here we used fluorescein ET-1 and 125I-ET-1 to provide evidence for ET-1 receptors in cardiac transverse tubules (T-tubules). Moreover, the ETA receptor and downstream effector phospholipase C-beta 1 were co-localized within T-tubules using standard immunofluorescence techniques, and protein kinase C (PKC)-epsilon-enhanced green fluorescent protein bound reversibly to T-tubules upon activation. Localized photorelease of diacylglycerol further suggested compartmentation of PKC signaling, with release at the myocyte "surface" mimicking the negative inotropic effects of bath-applied PKC activators and "deep" release mimicking the positive inotropic effect of ET-1. The functional significance of T-tubular ET-1 receptors was further tested by rendering the T-tubule lumen inaccessible to bath-applied ET-1. Such "detubulated" cardiac myocytes showed no positive inotropic response to 20 nM ET-1, despite retaining both a nearly normal twitch response to field stimulation and a robust positive inotropic response to 20 nm isoproterenol. We propose that ET-1 enhances myocyte contractility by activating ETA receptor-phospholipase C-beta 1-PKC-epsilon signaling complexes preferentially localized in cardiac T-tubules. Compartmentation of ET-1 signaling complexes may explain the discordant effects of ET-1 versus bath applied PKC activators and may contribute to both the specificity and diversity of the cardiac actions of ET-1.     

Effects of endothelin on adrenomedullin secretion and expression of adrenomedullin receptors in rat cardiomyocytes

Both endothelin (ET) and adrenomedullin (AM), produced by cardiac myocytes, are thought to be locally-acting hormones in the heart. Recently, calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) have been shown to function together to serve as AM receptors stimulating cAMP production. In the present study, we examined the effects of ET on AM secretion, intracellular cAMP response to AM, and gene expressions of CRLR and RAMPs in cultured cardiac myocytes. Synthetic ET-1 dose-dependently increased AM secretion from the cardiomyocytes. AM increased the intracellular cAMP level in a dose-dependent manner and the cAMP accumulation by AM was significantly amplified by 24 h preincubation with ET-1. 10 nmol/L ET-1 significantly increased the CRLR mRNA level without any effect on RAMP1 mRNA. 1 micromol/L ET-1 significantly reduced the RAMP2 mRNA level, but ET-1 dose-dependently increased the RAMP3 mRNA level in the cardiac myocytes. These findings suggest that ET-1 not only stimulates AM secretion, but also modulates intracellular cAMP responses to AM probably by altering the expressions of CRLR and RAMPs in rat cardiomyocytes.     

Regulation of ACE2 in cardiac myocytes and fibroblasts

Angiotensin-converting enzyme 2 (ACE2) preferentially forms angiotensin-(1-7) [ANG-(1-7)] from ANG II. We showed that cardiac ACE2 is elevated following treatment of coronary artery-ligated rats with AT1 receptor blockers (ARBs). Cardiac myocytes and fibroblasts were isolated from neonatal rats to determine the molecular mechanisms for the ACE2 upregulation by ARB treatment. ANG II significantly reduced ACE2 activity and downregulated ACE2 mRNA in cardiac myocytes, effects blocked by the ARB losartan, indicating that ANG II regulates ACE2. ANG II also reduced ACE2 mRNA in cardiac fibroblasts; however, no enzyme activity was detected, reflecting the limited expression of ACE2 in these cells. Endothelin-1 (ET-1) also significantly reduced myocyte ACE2 mRNA. The reduction in ACE2 mRNA by ANG II or ET-1 was blocked by inhibitors of mitogen-activated protein kinase kinase 1, suggesting that ANG II or ET-1 activates extracellular signal-regulated kinase (ERK) 1/ERK2 to reduce ACE2. Although ACE2 mRNA was not affected by ANG-(1-7), both the ANG II- and ET-1-mediated reductions in ACE2 mRNA were blocked by the heptapeptide. The ANG-(1-7) modulatory effect was prevented by the ANG-(1-7) receptor antagonist [D-Ala7]-ANG-(1-7), indicating that the ANG-(1-7) response was mediated by a specific AT(1-7) receptor. Myocyte treatment with atrial natriuretic peptide (ANP) also reversed the ACE2 mRNA downregulation by ANG II or ET-1, whereas treatment with ANP alone was ineffective. These results indicate that multiple hypertrophic and anti-hypertropic peptides regulate ACE2 production in myocytes, suggesting that ACE2 expression in the heart is dependent upon the compliment and concentration of regulatory molecules.     

PKC-ε mediates multiple endothelin-1 actions on systolic Ca2+ and contractility in ventricular myocytes

Endothelin-1 (ET-1) induces positive inotropy (enhanced contractility) in cardiac muscle, but establishing underlying cellular mechanisms has been controversial in part because of a growing number of signaling pathways and end effectors targeted by ET-1. Here we present evidence that ET-1 induces positive inotropism in ventricular tissue by increasing both systolic Ca2+ and myofilament Ca2+ sensitivity. To examine the roles of PKC-δ and PKC-ε in these acute responses to ET-1, kinase inactive dominant negative PKC (dn-PKC) constructs were expressed in adult rat ventricular myocytes. Yellow fluorescent protein (YFP) was fused to dn-PKC constructs to visualize expression and localization of dn-PKC in living myocytes. Due to an alanine to glutamate mutation in the pseudosubstrate site, dn-PKCs constitutively translocated to anchoring sites and were unaffected by agonist or phorbol ester treatment. Dn-PKC-δ-YFP mainly distributed at Z-lines and at intercalated disks in adult myocytes, whereas dn-PKC-ε-YFP stained the surface sarcolemma, T-tubules/Z-lines and perinuclear region. Myocytes expressing dn-PKC-δ-YFP showed normal systolic Ca2+ and contractile responses to ET-1. In contrast, the entire ensemble of ET-1 responses was blocked in myocytes expressing dn-PKC-ε-YFP including increased Ca2+ transients, enhanced myofilament Ca2+ sensitivity, and positive inotropy. This report provides direct evidence that PKC-ε is activated early and robustly following ET-1 stimulation and thus mediates multiple intracellular changes underlying the acute actions of ET-1 on myocardium.     

Endothelin inhibits adenylate cyclase and stimulates phosphoinositide hydrolysis in adult cardiac myocytes.

We have assessed the effects of endothelin-1 (ET-1) on transmembrane signaling in adult rat ventricular myocytes. ET-1 stimulates phosphoinositide hydrolysis with an EC50 of 0.3-0.8 nM. This stimulation is linear for up to 30 min in the presence of a protease inhibitor, is additive with the effects of other stimulators of phosphoinositide hydrolysis, is not inhibited by the Ca2+ entry blocker, nifedipine, and is insensitive to pertussis toxin. ET-1 also reduces cyclic AMP production in myocytes in response to isoproterenol and forskolin (EC50, 1 nM). This cyclic AMP-lowering effect of ET-1 is sensitive to pertussis toxin, can be demonstrated directly in assays of adenylate cyclase activity of myocyte membranes, and seems to be mediated by Gi. These data indicate that the effects of endothelin on adult cardiac myocytes involve multiple signaling pathways, including enhanced activity of the inositol phosphate pathway and a decrease in cyclic AMP-mediated responses, neither of which seems likely to account for the positive contractile effects of endothelin.     

Activation of peroxisome proliferator-activated receptor-alpha prevents glycogen synthase 3beta phosphorylation and inhibits cardiac hypertrophy

Activation of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) has been recently reported to inhibit vascular inflammatory response and prevent cardiac hypertrophy. However, it is unclear how the activation of PPAR-alpha regulates hypertrophic response. In the present study, we found that application of fenofibrate and overexpression of PPAR-alpha inhibited endothelin-1 (ET-1)-induced phosphorylation of protein kinase B (Akt) at Ser473 and glycogen synthase kinase3beta (GSK3beta) at Ser9, and prevented ET-1-induced nuclear translocation of NFATc4 in cardiomyocytes. Moreover, co-immunoprecipitation studies showed that fenofibrate strongly induced the association of nuclear factor of activated T cells (NFATc4) with PPAR-alpha. These results suggest that activation of PPAR-alpha inhibits ET-1-induced cardiac hypertrophy through regulating PI3K/Akt/GSK3beta and NFAT signaling pathways.     

Peroxisome proliferator-activated receptor-gamma activators inhibit IFN-gamma-induced expression of the T cell-active CXC chemokines IP-10, Mig, and I-TAC in human endothelial cells

Peroxisome proliferator-activated receptor-gamma (PPARgamma), a member of the nuclear hormone receptor superfamily originally shown to play an important role in adipocyte differentiation and glucose homeostasis, is now known to regulate inflammatory responses. Given the importance of endothelial cell (EC)-derived chemokines in regulating leukocyte function and trafficking, we studied the effects of PPARgamma ligands on the expression of chemokines induced in ECs by the Th1 cytokine IFN-gamma. Treatment of ECs with PPARgamma activators significantly inhibited IFN-gamma-induced mRNA and protein expression of the CXC chemokines IFN-inducible protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), whereas expression of the CC chemokine monocyte chemoattractant protein-1 was not altered. PPARgamma activators decreased IFN-inducible protein of 10 kDa promoter activity and inhibited protein binding to the two NF-kappaB sites but not to the IFN-stimulated response element ISRE site. Furthermore, PPARgamma ligands inhibited the release of chemotactic activity for CXC chemokine receptor 3 (CXCR3)-transfected lymphocytes from IFN-gamma-stimulated ECs. These data suggest that anti-diabetic PPARgamma activators might attenuate the recruitment of activated T cells at sites of Th1-mediated inflammation.     

PPARα activation inhibits endothelin-1-induced cardiomyocyte hypertrophy by prevention of NFATc4 binding to GATA-4

Peroxisome proliferator-activated receptor alpha (PPARα) has been implicated in the pathogenesis of cardiac hypertrophy, although its mechanism of action remains largely unknown. To determine the effect of PPARα activation on endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy and explore its molecular mechanisms, we evaluated the interaction of PPARα with nuclear factor of activated T-cells c4 (NFATc4) in nuclei of cardiomyocytes from neonatal rats in primary culture. In ET-1-stimulated cardiomyocytes, data from electrophoretic mobility-shift assays (EMSA) and co-immunoprecipitation (co-IP) revealed that fenofibrate (Fen), a PPARα activator, in a concentration-dependent manner, enhanced the association of NFATc4 with PPARα and decreased its interaction with GATA-4, in promoter complexes involved in activation of the rat brain natriuretic peptide (rBNP) gene. Effects of PPARα overexpression were similar to those of its activation by Fen. PPARα depletion by small interfering RNA abolished inhibitory effects of Fen on NFATc4 binding to GATA-4 and the rBNP DNA. Quantitative RT-PCR and confocal microscopy confirmed inhibitory effects of PPARα activation on elevation of rBNP mRNA levels and ET-1-induced cardiomyocyte hypertrophy. Our results suggest that activated PPARα can compete with GATA-4 binding to NFATc4, thereby decreasing transactivation of NFATc4, and interfering with ET-1 induced cardiomyocyte hypertrophy.     

Time course alterations of myocardial endothelin-1 production during the formation of exercise training-induced cardiac hypertrophy

Endothelin (ET)-1 is produced by endothelial cells and cardiac myocytes. ET-1 has positive inotropic and chronotropic effects on the heart and causes myocardial cell hypertrophy. Exercise training induces a physiologic cardiac hypertrophy. To study whether myocardial ET-1 is involved in the formation of exercise training-induced cardiac hypertrophy, we investigated time-course alterations of myocardial ET-1 gene expression and ET-1 peptide level in the heart of rats during a formative process of exercise training-induced cardiac hypertrophy. We used the hearts of rats that had been exercise-trained for 4 weeks (4WT) or 8 weeks (8WT) and sedentary control rats for 4 weeks (4WC) or 8 weeks (8WC). Exercise-trained rats performed treadmill running for 5 days/week (60 mins/day). Left ventricular mass index and wall thickness and stroke volume index, measured using echocardiography, in the 8WT group were significantly greater than in the 8WC group, although there were no differences between the 4WC and 4WT groups in these parameters. These results indicated that the 8WT rats developed physiologic cardiac hypertrophy, whereas the 4WT rats did not yet have cardiac hypertrophy. Myocardial ET-1 gene expression and tissue ET-1 concentration in the heart were significantly higher in the 8WT group than in the 8WC group, whereas these values did not differ between the 4WC and 4WT groups. The present study suggests that an alternation of myocardial ET-1 production corresponds with the formation of exercise training-induced cardiac hypertrophy. Therefore, the exercise training-induced change in myocardial ET-1 production may participate in a mechanism of exercise training-induced cardiac adaptation (e.g., cardiac hypertrophy).     

Endothelin-1 receptor subtypes expression and binding in a perfused rat model of myocardial infarction

Endothelin-1 (ET-1) pathophysiologic actions are mediated via binding with two receptor subtypes, ET(A) and ET(B). Release of ET-1 from endocardial endothelial cells and cardiac myocytes can modulate heart tissue necrosis and alterations. This study investigates the remodeling processes in Sprague-Dawley rats of myocardial infarction (MI) induced by ligating the left anterior descending coronary artery. Histological studies were done on cell type distribution using cell specific markers and Western blot analysis to localize ET-1 receptor subtypes and assess their expression post-MI. In addition, the binding kinetics of ET-1 with its receptors in heart perfusion, inlet via the aortic lumen and effluent outlet via the right atrium, between two animal model-subgroups were done: (1) sham-operated, and sham-operated-CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate)-treated; and (2) MI-operated, and MI-operated-CHAPS-treated. Effluent ET-1 concentration was plotted vs. time using a physical model for 1:1 ligand-receptor binding at coronary endothelium and myocytes. First order impulse function was used to calculate the affinity constants. In MI hearts, fluorescence activity increased for ET(A) vs. ET(B) across areas of the muscle compared to normal hearts. Western blotting showed upregulation of ET(A) and ET(B) receptors in MI compared with normal hearts. Results of ET-1 binding affinity post-MI indicated drastic reduction in spite the upregulation of ET(B) on coronary endothelium. Furthermore, substantial affinity increase was observed between ET-1 binding with ET(A) at the myocyte site. These findings stipulate that during 1 month post-MI some biochemical and hormonal effects could alter ET-1 receptor subtype(s) regulation and pharmacodynamics thus predisposing to cardiac hypertrophy and mitogenesis.     

Ca2+/calmodulin-dependent kinase II and calcineurin play critical roles in endothelin-1-induced cardiomyocyte hypertrophy

Endothelin-1 (ET-1) induces cardiac hypertrophy. Because Ca(2+) is a major second messenger of ET-1, the role of Ca(2+) in ET-1-induced hypertrophic responses in cultured cardiac myocytes of neonatal rats was examined. ET-1 activated the promoter of the beta-type myosin heavy chain gene (beta-MHC) (-354 to +34 base pairs) by about 4-fold. This activation was inhibited by chelation of Ca(2+) and the blocking of protein kinase C activity. Similarly, the beta-MHC promoter was activated by Ca(2+) ionophores and a protein kinase C activator. beta-MHC promoter activation induced by ET-1 was suppressed by pretreatment with the calmodulin inhibitor, W7, the Ca(2+)/calmodulin-dependent kinase II (CaMKII) inhibitor, KN62, and the calcineurin inhibitor, cyclosporin A. beta-MHC promoter activation by ET-1 was also attenuated by overexpression of dominant-negative mutants of CaMKII and calcineurin. ET-1 increased the activity of CaMKII and calcineurin in cardiac myocytes. Pretreatment with KN62 and cyclosporin A strongly suppressed ET-1-induced increases in [(3)H]phenylalanine uptake and in cell size. These results suggest that Ca(2+) plays a critical role in ET-1-induced cardiomyocyte hypertrophy by activating CaMKII- and calcineurin-dependent pathways.     

Effects of chronic endothelin-1 stimulation on cardiac myocyte contractile function

Endothelin-1 (ET-1) has acute positive inotropic effects, but consequences of chronically increased ET-1 on contractile function of cardiac myocytes are largely unknown. In the present study, effects of long-term treatment with ET-1 (10 nM) for 5 days on both force development [force of contraction (FOC)] and kinetics of contraction were determined in heart tissue reconstituted from rat cardiac cells. Isometric force was measured in response to cumulative concentrations of Ca(2+) and isoprenaline. ET-1 augmented basal FOC by 64 +/- 11% (P < 0.05), which was associated with a significantly blunted contractile response to Ca(2+) and isoprenaline. Moreover, ET-1 significantly prolonged relaxation (62 +/- 3 vs. 53 +/- 2 ms). Selective ET(A) (BQ-123) and ET(B) receptor blockade (BQ-788) demonstrated that effects of ET-1 on contractile function were mediated through the ET(A) receptor subtype. Effects of ET-1 were prevented by cotreatment with either Ro31-8425, a PKC inhibitor, or dimethylamiloride, an inhibitor of the Na(+)/H(+) exchanger. In contrast to long-term ET-1 treatment, no changes in contractile parameters were observed after ET-1 treatment for 3 h before force measurement. These data suggest that chronic ET-1 stimulation has dual effects on contractility: improvement of basal force but impairment of twitch kinetics and inotropic responsiveness to beta-adrenoceptor stimulation. The signaling pathways involved include ET(A) receptors, PKC, and the Na(+)/H(+) exchanger. The present in vitro findings raise the possibility that ET-1 may exert both adaptive and maladaptive effects in the failing myocardium in which local accumulation of ET-1 is present.     

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。