Canine ventricular myocytes possess a renin-angiotensin system that is upregulated with heart failure.

Ventricular pacing leads to a dilated myopathy in which cell death and myocyte hypertrophy predominate. Because angiotensin II (Ang II) stimulates myocyte growth and triggers apoptosis, we tested whether canine myocytes express the components of the renin-angiotensin system (RAS) and whether the local RAS is upregulated with heart failure. p53 modulates transcription of angiotensinogen (Aogen) and AT(1) receptors in myocytes, raising the possibility that enhanced p53 function in the decompensated heart potentiates Ang II synthesis and Ang II-mediated responses. Therefore, the presence of mRNA transcripts for Aogen, renin, angiotensin-converting enzyme, chymase, and AT(1) and AT(2) receptors was evaluated by reverse transcriptase-polymerase chain reaction in myocytes. Changes in the protein expression of these genes were then determined by Western blot in myocytes from control dogs and dogs affected by congestive heart failure. p53 binding to the promoter of Aogen and AT(1) receptor was also determined. Ang II in myocytes was measured by ELISA and by immunocytochemistry and confocal microscopy. Myocytes expressed mRNAs for all the constituents of RAS, and heart failure was characterized by increased p53 DNA binding to Aogen and AT(1). Additionally, protein levels of Aogen, renin, cathepsin D, angiotensin-converting enzyme, and AT(1) were markedly increased in paced myocytes. Conversely, chymase and AT(2) proteins were not altered. Ang II quantity and labeling of myocytes increased significantly with cardiac decompensation. In conclusion, dog myocytes synthesize Ang II, and activation of p53 function with ventricular pacing upregulates the myocyte RAS and the generation and secretion of Ang II. Ang II may promote myocyte growth and death, contributing to the development of heart failure.

Evidence for a role of an intracardiac renin-angiotensin system in normal and failing hearts.

Although substantial evidence of a cardiac RAS has been obtained in the past decade, a number of important questions remain unanswered. These include identification and localization of the cell types responsible for production of the system's components as well as the regulation of synthesis, storage, and secretion pathways for each component. Future studies, which will utilize tools of molecular biology that have become recently available (for example, transgenic animal models), renin inhibitors, angiotensin receptor antagonists, and bradykinin antagonists, will help to elucidate specific roles of the cardiac RAS in normal and failing hearts.

Regulation of cytosolic calcium by angiotensins in vascular smooth muscle.

The carboxy terminal homologue of angiotensin II (Ang II), Ang-(3-8) or hexapeptide, was used as a model peptide to examine the types of receptor mechanisms involved in calcium mobilization in cultured vascular smooth muscle cells. Hexapeptide did not produce tachyphylaxis but did produce a sustained increase in intracellular calcium. Differences in the increase in intracellular calcium [( Ca2+]i) and the pattern of inositol phosphate production indicate that Ang-(3-8) and maximal concentrations of Ang II mobilize calcium through different mechanisms. The calcium-mobilizing mechanisms that predominate appear to depend on the concentration of angiotensin. Concentrations of Ang II greater than 10(-8) M produce sharp calcium transients in which the [Ca2+]i returns close to baseline within 1 minute after stimulation, but concentrations of Ang II equal to or less than 3 x 10(-9) M result in a plateau increase in calcium. Pretreatment with Bordetella pertussis toxin does not abolish either the calcium transient induced by Ang II or the plateau phase induced by Ang-(3-8), indicating that the GTP-transducing protein that couples the receptor to phospholipase C or, possibly, a receptor-operated calcium channel is not Bordetella pertussis toxin sensitive.

Cardiotrophin-1 increases angiotensinogen mRNA in rat cardiac myocytes through STAT3 : an autocrine loop for hypertrophy.

-Cardiotrophin-1, an interleukin-6-related cytokine, stimulates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and induces cardiac myocyte hypertrophy. In this study, we demonstrate that cardiotrophin-1 induces cardiac myocyte hypertrophy in part by upregulation of a local renin-angiotensin system through the JAK/STAT pathway. We found that cardiotrophin-1 increased angiotensinogen mRNA expression in cardiac myocytes via STAT3 activation. Tyrosine phosphorylation of STAT3 by cardiotrophin-1 treatment resulted in STAT3 homodimer binding to the St-domain in the angiotensinogen gene promoter, which lead to promoter activation in a transient transfection assay. Cardiotrophin-1-induced STAT3 tyrosine phosphorylation and binding to the St-domain were suppressed by AG490, a specific JAK2 inhibitor, which also attenuated cardiotrophin-1-stimulated angiotensinogen promoter activity. Cardiotrophin-1 did not activate the angiotensinogen gene promoter that contained a substitution mutation within the St-domain. Finally, losartan, an angiotensin II type 1 receptor antagonist, significantly attenuated cardiotrophin-1-induced hypertrophy of neonatal rat cardiac myocytes. Angiotensin II is known to induce cardiac myocyte hypertrophy by activating the G-protein-coupled angiotensin II type 1 receptor. Our results suggest that upregulation of angiotensinogen and angiotensin II production contribute to cardiotrophin-1-induced cardiac myocyte hypertrophy and emphasize an important interaction between G-protein-coupled and cytokine receptors.

Captopril modifies gene expression in hypertrophied and failing hearts of aged spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) exhibits a transition from stable compensated left ventricular (LV) hypertrophy to heart failure (HF) at a mean age of 21 months that is characterized by a decrease in alpha-myosin heavy chain (alpha-MHC) gene expression and increases in the expression of the atrial natriuretic factor (ANF), pro-alpha1(III) collagen, and transforming growth factor beta1 (TGF-beta1) genes. We tested the hypotheses that angiotensin-converting enzyme inhibition (ACEI) in SHR would prevent and reverse HF-associated changes in gene expression when administered prior to and after the onset of HF, respectively. We also investigated the effect of ACEI on circulating and cardiac components of the renin-angiotensin system. ACEI (captopril 2 g/L in the drinking water) was initiated at 12, 18, and 21 months of age in SHR without HF and in SHR with HF. Results were compared with those of age-matched normotensive Wistar-Kyoto (WKY) rats, and to untreated SHR with and without evidence of HF. ACEI initiated prior to failure prevented the changes in alpha-MHC, ANF, pro-alpha1(III) collagen, and TGF-beta1 gene expression that are associated with the transition to HF. ACEI initiated after the onset of HF lowered levels of TGF-beta1 mRNA by 50% (P<.05) and elevated levels of alpha-MHC mRNA two- to threefold (P<.05). Circulating levels of renin and angiotensin I were elevated four- to sixfold by ACEI, but surprisingly, plasma levels of angiotensin II were not reduced. ACEI increased LV renin mRNA levels in WKY and SHR by two- to threefold but did not influence LV levels of angiotensinogen mRNA. The results suggest that the anti-HF benefits of ACEI in SHR may be mediated, at least in part, by effects on the expression of specific genes, including those encoding alpha-MHC, ANF, TGF-beta1, pro-alpha1(III) collagen, and renin-angiotensin system components.

Sensitive bioassay for the detection and quantification of angiotensin II in tissue culture medium.

We have developed a sensitive, high-throughput bioassay to quantify angiotensin II in culture medium. Using Chinese hamster ovary cells that stably express a transfected angiotensin II receptor as target cells, we demonstrated that an agonist-stimulated myelin basic protein kinase response can be used as a basis of quantitative bioassay for angiotensin II. The assay permits detection of as little as 10 pg of angiotensin II in 1 mL of medium and offers an excellent alternative to HPLC and radioimmunoassay. This approach may also be applicable for quantification of other peptide hormones or growth factors produced by cell cultures.

Paracrine actions of cardiac fibroblasts on cardiomyocytes: implications for the cardiac renin-angiotensin system.

Conditioned medium of cardiac fibroblasts was found to induce protein synthesis and signal transduction events rapidly, and to increase angiotensinogen messenger RNA (mRNA) levels in neonatal rat ventricular myocytes. Within 4 hours, fibroblast-conditioned medium (FCM) stimulated protein synthesis in cardiac myocytes, independent of the contractile state, and induced marked increases within 24 hours in total protein content. Endothelin- released by cardiac fibroblasts was not responsible for the stimulation of protein synthesis. FCM rapidly activated signal transduction events in cardiac myocytes associated with hypertrophic stimuli, including: (1) increased tyrosine phosphorylation of several prominent protein bands; (2) mitogen-activated protein kinases (ERK 1 and ERK 2); and (3) protein kinase C. Finally, FCM caused an increase at 8 hours in angiotensinogen mRNA levels of cardiac myocytes, whereas no effect was observed on mRNA levels for renin or the type 1 angiotensin II receptor (AT1). Our results suggest that cardiac fibroblasts produce a factor that rapidly activates cardiac myocyte growth through a membrane receptor that couples to conventional signal transduction pathways.

Angiotensin II stimulation of left ventricular hypertrophy in adult rat heart. Mediation by the AT1 receptor.

Angiotensin II (AII) has been reported to have direct hypertrophic actions in mammalian and avian myocardium. In this study we determined whether AII had receptor-mediated effects on stimulating cardiac hypertrophy independent of mechanical stimuli (increased cardiac afterload) in adult rats. Angiotensin II was infused into Sprague-Dawley rats for 7 and 14 days. Following this infusion, left ventricular mass indexed to body weight (LV/BW) increased 18.6 and 17.3%, respectively, compared with control (saline infused) rats. Administration of the nonpeptide AII receptor antagonist Dup 753 prevented the increase in left ventricular hypertrophy. Blockade of converting enzyme with enalapril maleate and treatment with a vasodilator had no effect on the AII-induced hypertrophy. In this animal model, the cardiac hypertrophy appeared to be independent of cardiac afterload, because normalization of blood pressure with hydralazine did not prevent the AII-induced hypertrophy. These in vivo studies indicate that AII-induced cardiac hypertrophy is mediated through AT1 angiotensin receptors.

The cardiac renin-angiotensin system: novel signaling mechanisms related to cardiac growth and function.

Angiotensin II, the effector peptide of the renin-angiotensin system, has been demonstrated to be involved in the regulation of cellular growth of several tissues in response to developmental, physiological, and pathological processes. The recent identification of renin-angiotensin system components and localization of angiotensin II receptors in cardiac tissue suggests that locally synthesized Ang II can modulate functional and growth responses in cardiac tissue. In this review, regulation of the cardiac RAS is discussed, with an emphasis on growth-related Ang II signal transduction systems.

Effects of captopril and bradykinin on arterial pressure in rats with mestranol-induced hypertension.

Hypertension was produced in 20 female rats by the oral administration of mestranol for 6 months; 20 control rats were not given mestranol during this time. The i.v. infusion of the angiotensin converting enzyme inhibitor, captopril, into 10 conscious mestranol-treated rats reduced mean arterial pressure from 139 +/- 2 (SEM) to 130 +/- 2 mm Hg (P less than 0.01); mean arterial pressure in 10 conscious control rats averaged 123 +/- 3 mm Hg, and was not significantly changed during captopril infusion. Injections of bradykinin at 0.1, 0.3, and 1.0 microgram/100 gm body weight decreased mean arterial pressure significantly more in 10 mestranol-treated rats than in 10 control rats. These results suggest that the ability of captopril to lower arterial pressure in rats with mestranol-induced hypertension may be due, at least in part, to the action of captopril to enhance bradykinin.

Stretch-induced MAP kinase activation in cardiac myocytes: differential regulation through beta1-integrin and focal adhesion kinase.

Mitogen-activated protein (MAP) kinases have been implicated in hemodynamic load induced heart failure. Both angiotensin II (Ang II) and mechanical stretch activate MAP kinases in cardiac myocytes. In this study, we used a neonatal rat ventricular myocyte (NRVM) model to determine the role of focal-adhesion kinase (FAK) in beta1 integrin mediated MAP kinase activation in response to mechanical stretch in presence and absence of Ang II receptor blockade (ATB). NRVM plated on deformable membranes coated with collagen IV were exposed to 20% equiaxial static-stretch. beta1 integrin signaling was blocked by adenovirus-mediated expression of a dominant-negative form of beta1D integrin (tac-beta1D). FAK signaling was disrupted by infecting NRVM with adenovirus expressing FAK-related non-kinase (FRNK). Western blot analysis was used to assess the phosphorylation of MAP kinases. In the presence and absence of ATB, mechanical stretch caused maximal phosphorylation of ERK, p38 and JNK at 5 min, which was significantly attenuated in NRVM expressing tac-beta1D. In the presence of ATB, FRNK overexpression significantly increased basal phosphorylation of ERK (40.2+/-8.6% P<0.05), p38 (39.5+/-11.7%, P<0.05), JNK (86+/-29.4%, P<0.05) and stretch-induced p38 (48.1+/-8.7%, P<0.05) and JNK (85.0+/-19.4%, P<0.05) phosphorylation. However, in the absence of ATB, FRNK overexpression significantly reduced basal and stretch-induced phosphorylation of only ERK. Examination of FAK activation revealed that beta1 integrin was required for stretch-induced phosphorylation of FAK at Y397 and Y925, but not Y861. In summary, mechanical stretch-activated ERK1/2, p38 and JNK through FAK independent and dependent mechanisms. Beta1 integrin was required for FAK independent activation of all three MAP kinases, whereas cross-talk between beta1 integrin and Ang II receptors mediated FAK dependent regulation of ERK1/2.

The cardiac renin-angiotensin system: conceptual, or a regulator of cardiac function?

Angiotensin II, the effector peptide of the renin-angiotensin system, regulates cellular growth in response to developmental, physiological, and pathological processes. The identification of renin-angiotensin system components and angiotensin II receptors in cardiac tissue suggests the existence of an autocrine/paracrine system that has effects independent of angiotensin II derived from the circulatory system. To be functional, a local renin-angiotensin system should produce sufficient amounts of the autocrine and/or paracrine factor to elicit biological responses, contain the final effector (angiotensin II receptor), and respond to humoral, neural, and/or mechanical stimuli. In this review, we discuss evidence for a functional cardiac renin-angiotensin system.

The angiotensin II receptor and the actions of angiotensin II.

Angiotensin II (Ang II) is a potent effector peptide of the renin-angiotensin system that exerts a wide variety of physiological actions on the cardiovascular, renal, endocrine, and central and peripheral nervous systems. Angiotensin exerts its actions by binding to specific receptors in the plasma membrane of various tissues. Structure-activity relationship studies and competition-binding experiments have identified a potency series of angiotensin analogues. Such studies have demonstrated that target organs display different preferences for Ang II and homologues such as Ang III and des-[Phe8] angiotensin II. Similarly, agents that normally are considered to be pure receptor antagonists for a given response (tissue) are full agonists in other tissues. Indirect evidence obtained from the above studies have led to the speculation that there are multiple angiotensin receptor subtypes among various tissues as well as within single cell types. Multiple mechanisms of signal transduction have been demonstrated for angiotensin. For example, depending on the effector organ, angiotensin stimulates phosphoinositide turnover and release of internal calcium, modulates voltage-dependent calcium channels, directly activates calcium channels, and inhibits adenylate cyclase activity. Recently, the identification of selective, high-affinity peptide and nonpeptide antagonists has resulted in further characterization of angiotensin receptors into distinct subtypes. In addition, dithiothreitol, an agent that reduces disulfide bridges, has been a useful tool in the characterization of angiotensin receptors as the subtypes apparently are not affected equally by this agent. However, further work needs to be performed to characterize angiotensin receptors with respect to heterogeneity, structure, transducing mechanisms, and physiological function.

Regulation of angiotensinogen gene expression and protein in neonatal rat cardiac fibroblasts by glucocorticoid and beta-adrenergic stimulation.

We previously demonstrated the presence of components for a renin-angiotensin system in fibroblasts cultured from neonatal rat ventricles, the regulation of expression of which has not been studied. Since glucocorticoids and beta-adrenergic stimuli have been implicated in cardiac hypertrophy, and function as regulators of the circulating renin-angiotensin system, we examined the effects of dexamethasone and isoproterenol on angiotensinogen mRNA levels and protein secretion in cultured neonatal rat cardiac fibroblasts. Treatment of cardiac fibroblasts for 8 h with 10 micromol/l isoproterenol or 100 nmol/l dexamethasone increased angiotensinogen mRNA levels by 246 +/- 7% and 1406 +/- 207%, respectively. Over 24 h, dexamethasone and isoproterenol increased angiotensinogen secretion by 148 +/- 32% and 123 +/- 26%, respectively. Angiotensin II, which has been reported to be a positive regulator of angiotensinogen synthesis and secretion in liver, markedly attenuated the effects of dexamethasone and isoproterenol on angiotensinogen mRNA expression and secretion. In the presence of 1 micromol/l angiotensin II, the stimulation in angiotensinogen secretion observed with dexamethasone and isoproterenol was decreased by 62% and 76%, respectively. The negative feedback of angiotensin II on angiotensinogen expression was primarily mediated through the type one angiotensin II (AT1) receptor (IC50 = 0.30 +/- 0.02 nmol/l). In summary, results from this study demonstrate that angiotensinogen mRNA levels and protein secretion in cardiac fibroblasts are positively regulated by glucocorticoid and beta-adrenergic stimulation. In addition, angiotensinogen production by cardiac fibroblasts is under negative feedback control of angiotensin II.

Cardiac actions of angiotensin II: Role of an intracardiac renin-angiotensin system.

The renin-angiotensin system has a varied role in the regulation of cardiac function, ranging from early receptor-mediated effects such as second messenger generation, to more delayed responses such as protein synthesis and cell growth. Clinically, the importance of the RAS in cardiovascular disease is becoming increasingly evident with the use of ACE inhibitors in treating various pathological processes. With evidence for the existence of a local RAS in the heart, the molecular and biochemical regulation of this system requires investigation. Much additional work needs to be directed toward elucidating the mechanisms by which the AII-receptor couples to cardiac growth, how the local RAS is regulated, and the nature of controls that modulate cardiac production and actions of this peptide. Increased understanding of the mechanisms by which AII actions are affected in cardiac tissue will likely lead to enhanced therapeutic modalities for the treatment of pathological cardiovascular conditions in which the RAS plays an integral role.

An improved method for absolute quantification of mRNA using multiplex polymerase chain reaction: determination of renin and angiotensinogen mRNA levels in various tissues.

We have developed a multiplex, competitive, reverse-transcriptase polymerase chain reaction (RT-PCR) method which measures absolute levels of renin, angiotensinogen, and the housekeeping transcript elongation factor-1 alpha (EF-1 alpha) mRNA. Sample RNA was simultaneously titrated with serial dilutions of renin, angiotensinogen, and EF-1 alpha competitor RNAs which flanked the endogenous concentrations of target transcripts. The samples were coreverse transcribed in the presence of random primers and resulting first-strand cDNA was coamplified for 10-15 cycles with [32P]-dCTP and primers for renin angiotensinogen, after which EF-1 alpha primers were added. Amplified DNA was separated by electrophoresis on polyacrylamide gel and radioactivity in the bands was quantified by direct radioanalytical scanning. Three conditions were necessary to obtain absolute quantification of renin and angiotensinogen mRNA levels: (a) exogenous competitor RNA was used to control for tube-to-tube variability in the efficiencies of reverse transcription and amplification; (b) Sample RNA was titrated with flanking concentrations of competitor RNA to correct for intraassay differences in the efficiency of amplification due to concentration differences between competitor and target templates; and (c) a housekeeping transcript EF-1 alpha was used to control for tube-to-tube differences in RNA loading and/or degradation. We show that the multiplex RT-PCR method is precise and accurate over approximately three logs of transcript concentration and sensitive to less than 5 and 0.5 fg for renin and angiotensinogen mRNA, respectively. This method will be useful for absolute quantification of target mRNAs, especially when the amount of sample RNA is limited or unknown and/or the gene expression is low.

Angiotensin II signalling pathways in cardiac fibroblasts: conventional versus novel mechanisms in mediating cardiac growth and function.

Angiotensin II has been demonstrated to be involved in the regulation of cellular growth of several tissues in response to developmental, physiological, and pathophysiological processes. Angiotensin II has been implicated in the developmental growth of the left ventricle in the neonate and remodeling of the heart following chronic hypertension and myocardial infarction. The inhibition of DNA synthesis and collagen deposition in myocardial interstitium following myocardial infarction by angiotensin converting enzyme inhibitor, suggests that angiotensin II mediates interstitial and perivascular fibrobrosis by preventing fibroblast proliferation. In the past, little attention was focused on the identity and functional roles of cardiac fibroblasts. Recent in vitro studies utilizing cultured cardiac fibroblasts demonstrate that angiotensin II, acting via the AT1 receptor, initiates intracellular signalling pathways in common with those of peptide growth factors. Below, we describe growth-related aspects of cardiac fibroblasts with respect to angiotensin II receptors, conventional and novel signal transduction systems, secretion of extracellular matrix proteins and growth factors, and localization of renin-angiotensin system components.

Angiotensin III and pressor responsiveness in 3-day renal artery stenosis rabbits.

This study examined the ability of [des-Asp1]angiotensin II (angiotensin III, ANG III) to interact with the angiotensin II (ANG II) receptors involved in pressor hyperresponsiveness in one-kidney, 3-day renal artery stenosis (RAS) rabbits. In experiment 1, six 3-day RAS rabbits had significantly larger pressor responses to norepinephrine (NE; 800 ng.min-1.kg body wt-1) than did six 3-day controls. The intravenous infusion of captopril, an angiotensin-converting enzyme inhibitor, diminished the pressor response NE in the RAS rabbits. ANG III at 0.5, 1.0, and 2.0 pmol.min-1.kg-1 iv with captopril resulted in a progressive restoration of pressor hyperresponsiveness to NE in the RAS rabbits, and ANG II at 2.0 pmol.min-1.kg-1 iv produced no further enhancement of the pressor responses to NE. In experiment 2, 12 3-day RAS rabbits had pressor hyperresponsiveness to NE that was alleviated by the infusion of captopril and was restored by the intravenous infusion of ANG III (2 pmol.min-1.kg-1). In six of the these rabbits an additional intravenous infusion of the ANG II antagonist [Sar1,Ile8]ANG II at 300 ng.min-1.kg body wt-1 diminished the pressor hyperresponsiveness to NE; in the other six rabbits the infusion of [Sar1,Ala8] ANG II at 6 micrograms.min-1.kg body wt-1 did not reduce the pressor hyperresponse to NE. In experiment 3, the infusion of ANG III (5 pmol.min-1.kg-1) in six normal rabbits did not alter the pressor responses to NE (400 ng.min-1.kg-1), whereas the infusion of ANG II (5 pmol.min-1.kg-1) resulted in increased pressor responses to NE.(ABSTRACT TRUNCATED AT 250 WORDS)

Involvement of protein kianse C and Ca2+ in angiotensin II-induced mitogenesis of cardiac fibroblasts.

Angiotensin (ANG) II has been previously shown to stimulate proliferation of neonatal rat cardiac fibroblasts via AT1 receptors. Here we conducted studies to assess involvement in this process of two second messengers linked to AT1 receptors, protein kinase C (PKC) and Ca2+. Several findings argue against a dominant role for PKC in ANG II-induced mitogenesis: 1) [Sar1]ANG II, which produced a modest, transient increase in PKC activity, was equally effective in inducing thymidine incorporation into DNA in PKC-depleted cells, whereas the effect of platelet-derived growth factor (PDGF)-BB on thymidine incorporation was reduced to the level observed with [Sar1]ANG II; 2) phorbol 12-myristate 13-acetate (PMA), a potent PKC stimulator, was ineffective in stimulating thymidine incorporation; and 3) PKC downregulation or the highly specific PKC inhibitor, compound 3, eliminated PMA-induced mitogen-activated protein (MAP) kinase activity but did not affect comparable increases induced by [Sar1]ANG II or PDGF-BB. Increased intracellular Ca2+ may be sufficient to account for [Sar1]ANG II-induced MAP kinase activity because ionomycin also increased MAP kinase activity and chelation of intracellular Ca2+ eliminated [Sar1]ANG II-induced activity in PKC-depleted fibroblasts. However, Ca2+ chelation did not prevent [Sar1]ANG II-induced MAP kinase activity in non-PKC-depleted fibroblasts. Thus ANG II can activate MAP kinase in cardiac fibroblasts by either Ca(2+)- or PKC-dependent pathways, and whereas the full effect of PDGF-BB on thymidine incorporation and cell proliferation requires a phorbol ester-sensitive PKC, the hyperplastic growth effect of ANG II does not.