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Effect of extracellular and intracellular angiotensins on heart cell function; on the cardiac renin–angiotensin system
Regulatory Peptides 114 (2003) 87 – 90 www.elsevier.com/locate/regpep
Invited review
Effect of extracellular and intracellular angiotensins on heart cell function; on the cardiac renin–angiotensin system Walmor De Mello * Department of Pharmacology, School of Medicine, Medical Sciences Campus, UPR, PO Box 365067, San Juan 00936-5067, PR, USA Received 12 September 2002; received in revised form 31 March 2003; accepted 5 April 2003
Abstract In this manuscript, I presented up-to-date evidence that intracellular and extracellular angiotensins have an important regulatory effect on the processes of heart cell communication and inward calcium current and that aldosterone modulates the effect of angiotensin II (Ang II) on the electrical properties of the heart. Moreover, I discussed the most relevant information about the origin of cardiac renin, the presence of a cardiac renin – angiotensin aldosterone system and its possible relevance for heart cell physiology and pathology. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Intracellular; Extracellular; Intracrine; Angiotensins; Renin; Aldosterone
The major function of cardiac muscle is contraction which propels the blood through systemic and pulmonary circulation. However, we must not forget that the heart is an outstanding electrochemical machine in which the energy stored by the membrane potential is periodically released, permitting the coupling between electrical and mechanical processes. Not only the ionic currents flowing through the surface cell membrane but the intercellular communication process mediated by gap junctions, are influenced by different factors including hormones. Indeed, evidence is available that angiotensin II (Ang II) regulates heart contractility, cell coupling, impulse propagation and is involved in the regulation of growth [1– 4]. These effects are mediated by Ang II receptors [5], which are 7-transmembrane domain receptors whose primary structures have been established by molecular cloning [6]. Exposure of AT1 receptors to Ang II is followed by translocation of the receptor to intracellular vesicles [7] and internalization of the Ang II AT1 receptor complex which occurs with a half-life of < 2 min [8,9]. The role of an internalized Ang II – receptor complex on heart cell function or as a source of intracellular Ang II merits further studies. The activation of AT2 receptors counteracts the AT1 receptor-induced progression of interstitial fibrosis and induces apoptosis [10] which is also induced by activation of AT1 receptors [11]. * Tel.: +1-787-766-4441; fax: +1-787-282-0568. E-mail address: [email protected] (W. De Mello).
It has been shown that Ang II added to the bath solution, reduces the gap junction conductance in cell pairs isolated from the ventricle of cardiomyopathic hamsters at an advanced stage of the disease [2,3]. This effect of Ang II, which appeared within seconds, was suppressed by losartan indicating that its effect was related to the activation of AT1 receptors. Moreover, the decline on cell coupling depends on the activation of protein kinase C [2,3]. Evidence is available that connexin 43, a major gap junction protein, is a MAP kinase substrate and that phosphorylation of Ser255, Ser279 and Ser282 initiates the downregulation of gap junction communication. Indeed, the levels of tyrosine phosphorylated connexin 43 are increased in the failing heart [12] supporting the idea that tyrosine phosphorylation is involved in the low values of junctional conductance found in the heart of cardiomyopathic hamsters [13]. Because it is known that tyrosine phosphorylation plays an important role on Ang II-mediated signal transduction [14], it is reasonable to think that it is also involved in the effect of the peptide on heart cell coupling. It has been found that AT1 receptor activation leads to an increased tyrosine phosphorylation of several intracellular substrates and that the effect of Ang II on remodeling is achieved through the activation of JAK –STAT pathways [15,16]. Moreover, c-Jun N-terminal kinase activation, a stress – activated protein kinase, mediates downregulation of connexin 43 in cardiomyocytes [17] but the relationship between this finding and the remodeling of gap junctions during pathological states needs to be clarified.
0167-0115/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(03)00121-6
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W. De Mello / Regulatory Peptides 114 (2003) 87–90
The cell uncoupling elicited by Ang II seen in the ventricle of cardiomyopathic hamsters, at an advanced stage of heart failure, shows that the activation of the plasma renin – angiotensin system induces slow conduction and reentry—two major causes of cardiac arrhythmias [18]. Moreover, the local application of Ang II to different spots of the failing ventricle showed an increase in action potential duration in some cells but not in other [18]. This means that the peptide elicits an increased dispersion of the action potential duration (and consequently of QRS)—a cause of cardiac arrhythmias [18]. The mechanism by which Ang II increments the action potential duration is not known but there is the possibility that it is related to a change in potassium conductance (see Ref. [19]). It has been shown that Ang II is a cardiovascular risk factor [20] and that ACE inhibitors like enalapril, increase heart cell coupling and impulse propagation, an effect in part related to the increment of the Na – K pump and membrane hyperpolarization [21,22].
1. Aldosterone modulates the effect of Ang II It is known that Ang II releases aldosterone from the adrenal gland. Therefore, it is important to know if aldosterone alters the effect of Ang II on heart muscle. Recently, it was found that the effect of Ang II on the electrical properties of adult rat heart is modulated by aldosterone [23]. Indeed, in rats treated with aldosterone for 48 h, Ang II, added to the extracellular medium, increases the action potential duration and refractoriness contrary to its effect in the controls in which both parameters are reduced by the same dose of Ang II [23]. This means that the arrhythmogenic action of Ang II in rat heart is reversed by aldosterone. Because the increment in action potential duration elicited by Ang II in aldosterone treated rats was suppressed by verapamil it is reasonable to conclude that it is related to an increased inward calcium current. Interestingly, the effect of isoproterenol on cardiac excitability and action potential duration was not altered by aldosterone [23]. These findings show, for the first time, that the effect of Ang II on the electrical and probably in other functions of the heart must be evaluated considering the presence or absence of aldosterone. Recently, it has been shown that aldosterone receptors are present in the heart [24] and that aldosterone is produced in the heart [25]. Chronic treatment with Ang II as well as a low sodium/high potassium diet, increased production of aldosterone [26,27] what raises the possibility that there is a cross-talk between angiotensin and aldosterone in the heart. Further studies are needed to confirm this hypothesis.
2. On the hypothesis of cardiac renin– angiotensin system There is evidence that there is a local renin –angiotensin system (RAS) in the heart, an idea based on the
demonstration that elements of the RAS cascade (renin, angiotensinogen, Ang I, Ang II and angiotensin converting enzyme (ACE)) are present in this tissue [28,29]. In normal heart, however, renin mRNA levels are extremely low or undetectable in nephrectomized animals [30] what signifies that the uptake of renin from plasma is the major source of cardiac renin [31]. The possibility exists that renin is internalized by mannose-6 phosphate receptors for which glycosylation of renin is needed [32] or that renin is bound to an unidentified protein [33,34] and that Ang II is synthesized inside the cells. The contribution of plasma renin to cardiac renin was tested using transgenic mice expressing human renin in the liver (TTRhRen-A3) which were mated to mice expressing human angiotensinogen exclusively in the heart (MHChAgt-2). These results indicated low or undetectable angiotensin peptide in the heart of single transgenic animals while double transgenic mice showed a remarkable increase of cardiac levels of Ang I and Ang II, indicating that plasma renin is able to act on its substract within the heart [35]. Recently, it has been shown that prorenin is internalized into cardiomyocytes independently of glycosylation and that this process is of functional significance because it leads to generation of angiotensins [36]. Other studies, however, indicate that prorenin induces myocyte proliferation, an effect unrelated to intracellular Ang II [37]. An important finding was that the heart expresses an alternative renin transcript (exon 1A renin) which is maintained intracellularly [38]. This expression, which is markedly enhanced after myocardial ischemia [38], supports the notion that there is an intracellular renin – angiotensin system [39]. Previous observations [40] showed that > 90% of cardiac Ang I and more than 75% of cardiac Ang II are synthesized at cardiac sites. Indeed, experimental evidence indicates that angiotensins are generated locally and released by the heart [41,42]. An important question is which factor or factors lead to activation of the cardiac renin – angiotensin system. Studies of Malhotra et al. [43] indicated that mechanical stretch increases the expression of renin and angiotensinogen genes in neonatal cardiac myocytes. Local mechanical stress rather then systemic factors seems to regulate the expression of these genes [44] because right ventricular hypertrophy, for instance, increases the mRNA levels of ACE, AT2 receptors and decreases the expression of AT1 receptors in the right ventricle but no change in expression of renin and angiotensinogen genes were found in the left ventricle [44]. In the failing heart of cardiomyopathic hamsters, the ACE activity is not increased at 2 months of age but it is appreciably enhanced afterwards when overt heart failure is seen [45]. Because aldosterone induces angiotensin converting enzyme gene expression in cultured cardiac cells [46], the question remains if aldosterone in involved in the activation of the cardiac renin – angiotensin system.
W. De Mello / Regulatory Peptides 114 (2003) 87–90
Previous studies from our laboratory indicated that intracellular renin caused a decrease of gap junction communication in heart muscle [47]. Furthermore, intracellular dialysis of Ang I into myocytes of cardiomyopathic hamsters, at an advanced stage of the disease, elicited total suppression of cell communication, an effect related to its conversion to Ang II because the effect of Ang I was greatly reduced by intracellular enalaprilat [2]. As a matter of fact, intracellular dialysis of Ang II into these cells abolished cell coupling, an effect suppressed by intracellular but not by extracellular losartan. These observations might indicate that the effect of intracellular Ang II is dependent upon the presence of an intracellular receptor similar to AT1. Evidence is available that there is a soluble angiotensinbinding receptor cytosolic protein with a molecular weight of 75 kDa [48 – 50]. Moreover, nuclear and chromatin Ang II receptors have been identified [51 – 53]. Immunochemical studies also indicated the presence of intracellular Ang II receptors in cardiomyocytes and fibroblasts [54]. This finding contrasts with previous studies of Kato et al. [55], which demonstrated that the porcine soluble angiotensinbinding protein is a microsomal endopeptidase—a finding that does not support the idea of an intracellular Ang II receptor. Intracellular injection of Ang II into ventricular cells of intact ventricle of cardiomyopathic hamsters, at 4 months of age, demonstrated an increase in action potential duration and refractoriness but cardiac arrhythmias appeared when larger amounts of Ang II (10 9 M) were injected (De Mello, in preparation). These changes elicited by intracellular Ang II, were suppressed by intracellular losartan (De Mello, in preparation). Additional experiments demonstrated that intracellular dialysis of Ang II reduces the inward calcium current in isolated cardiomyocytes of adult rats but increases it in myocytes of Golden hamsters [56], which suggests that intracellular Ang II is involved in the regulation of heart contractility, an effect that varies with the species. The increase of calcium current, which was related to the activation of protein kinase C [56], might be important for the failing heart. Indeed, Ang II dialyzed into ventriclar cells of cardiomyopathic hamsters cells, elicted an increment of the inward calcium current. Because the effect of intracellular Ang II on inward calcium current was not suppressed by losartan applied to the extracellular fluid, it is reasonable to think that the peptide is acting intracellularly (De Mello, unpublished). Transgenic animal models have been used to investigate the role of cardiac renin – angiotensin system. Overexpression of angiotensinogen gene in normal heart muscle cells caused an increment of the angiotensin II concentration in right and left ventricle and elicited hypertrophy of both ventricles without any change in arterial blood pressure [57,58]. These observations indicate that cardiac Ang II is able to cause ventricular hypertrophy independently of hemodynamic alterations.
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3. Conclusions The evidence gathered thus far supports the view that there is a cardiac renin – angiotensin system which is activated during pathological states such as heart failure. However, the debate continues about the site of Ang II synthesis. Extracellular and intracellular angiotensins play an important role on the control of heart cell function and aldosterone modulates the effect of Ang II on electrical properties of the heart. The role of intracellular angiotensins on cardiac cell function might indicate that therapeutic procedures must be developed to control intracellular angiotensin and renin levels. Further studies on the role of intracellular Ang II and aldosterone on cardiac functions will provide penetrating insights into the intricacies of the interactions between Ang II and aldosterone and their implications to heart cell biology.
Acknowledgements This work was supported by grants from the American Heart Association and NIH (HL-34148, 532943).
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[13] De Mello WC. Gap junctional conductance in cardiomyopathic hamsters. The role of c-src. Circ Res 1999;85:661 – 2. [14] Haendeler J, Berk B. Tyrosine phosphorylation is involved in Ang IImediated signal transduction. Regul Pept 2000;95:1 – 7. [15] Dostal DE, Hunt RA, Kule CE, Bhat GJ, Kakoor V, McWhinney CD, et al. Molecular mechanisms of angiotensin II in modulating cardiac function: intracellular effects and signal transduction pathways. J Mol Cardiol 1997;29:2893 – 902. [16] Kodama H, Fukuda K, Pan J, Makino S, Sano M, Takahashi T, et al. Biphasic activation of the JAK/STAT pathway by angiotensin II in rat cardiomyocytes. Circ Res 1998;82:244 – 50. [17] Petrich BG, Gong X, Lerner DL, Wang X, Brown JH, Saffitz JE, et al. c-Jun N-terminal kinase activation mediates downregulation of connexin 43 in cardiomyocytes. Circ Res 2002;91:640 – 7. [18] De Mello WC. Cardiac arrhythmias; the possible role of the renin angiotensin system. J Mol Med 2001;79:103 – 8. [19] Daleau P, Turgeon J. Angiotensin II modulates the delayed rectifier potassium current in guinea pig ventricular myocytes. Pflugers Arch 1994;427:553 – 5. [20] Gavras I, Gavras H. Angiotensin II as a cardiovascular risk factor. J Hum Hypertens 2002;16(Suppl 2):2 – 6. [21] De Mello WC. Electrical activity of the heart and angiotensin converting enzyme inhibitors; on the hyperpolarizing action of enalapril. J Hum Hypertens 2002;16(Suppl 1):89 – 92. [22] De Mello WC, Danser AHJ. Angiotensin II and the heart. On the intracrine renin – angiotensin system. Hypertension 2000;35:1183 – 8. [23] De Mello WC. Aldosterone modulates the effect of angiotensin II on the electrical properties of adult rat heart. J Cardiovasc Pharmacol 2002;40:90 – 5. [24] Bonvalet JP, Alfaidy N, Farman N, Lombes M. Aldosterone: intracellular receptors in human heart. Eur Heart J 1995;16(Suppl N):92 – 7. [25] Mizuno Y, Yoshimura M, Yasue H, Sakamoto T, Ogawa H, Kugiyama K, et al. Aldosterone production is activated in failing ventricles in humans. Circulation 2001;103:72 – 7. [26] Sivestre JS, Heymes C, Oubenaissa A, et al. Activation of cardiac aldosterone production in rat myocardial infarction. Circulation 1999; 99:2694 – 701. [27] Takeda Y, Takashi Y, Masashi D, et al. Cardiac aldosterone production in genetic hypertensive rats. Hypertension 2000;36:495 – 500. [28] Dzau VJ. Implications of local angiotensin production in cardiovascular physiology and pharmacology. Am J Cardiol 1987;59(Suppl A): 59A – 65A. [29] Jin M, Wilhelm MJ, Lang RE, Unger T, Lindpaintner K, Ganten D. Endogenous tissue renin – angiotensin systems; from molecular biology to therapy. Am J Med 1988;84(Suppl 3A):28 – 36. [30] Katz SA, Opsahl JA, Lunzer MM, Forbis LM, Hirsch AT. Effect of Bilateral nephrectomy on active renin, angiotensinogen and renin glycoforms in plasma and myocardium. Hypertension 1997;30:259 – 66. [31] Danser AHJ, van Katz JP, Admiraal PJJ, Derbx FHM, Lamers JMJ, Verdow PD, et al. Cardiac renin and angiotensins: uptake from plasma versus in situ synthesis. Hypertension 1994;24:37 – 48. [32] van Kesteren CAM, Danser AHJ, Derbx FHM, Dekkers DHW, Lamers JMJ, Saxena PR. Shalekamp MADH Mannose 6-phosphate receptor-mediated internalization and activation of prorenin by cardiac cells. Hypertension 1997;30:1389 – 96. [33] Campbell DJ, Valentin AJ. Identification of vascular renin-binding proteins by chemical cross-linking: inhibition of renin by renin inhibitors. J Hypertens 1994;12:879 – 90. [34] Sealey JE, Catanzaro DF, Lavin TN, Gahnem F, Pitarresi T, Hu LF, et al. Specific prorenin/renin binding (ProBP); identification and characterization of a novel membrane site. Am J Hypertens 1996;9:491 – 502. [35] Prescott G, Siversides DW, Chiu SML, Reudelhuber TL. Contribution of circulating renin to local synthesis of angiotensin peptides in the heart. Physiol Genomics 2000;4:67 – 73. [36] Peters J, Farrenkopf R, Clausmeyer S, Zimmer J, Kantachuverisi S, Sharp GFM, et al. Functional significance of prorenin internalization in the rat heart. Circ Res 2002;90:1135 – 41.
[37] Saris JJ, Mark MED, van den Eijnden J, Lamers MJ, Saxena PP, Marteen ADH, et al. Prorenin-induced myocyte proliferation. No role for intracellular angiotensin II. Hypertension 2002;39:573 – 7. [38] Peters J, Clausmeyer S. Intracellular sorting of renin:cell type specific differences and their consequences. J Mol Cell Cardiol 2002;34: 1561 – 8. [39] Cook JL, Zhang Z, Re RN. In vitro evidence for an intracellular site of angiotensin action. Circ Res 2001;89:1138 – 46. [40] van Katz JP, Danser AHJ, van Maegen J, Sassen LMA, Verdouw PD, Schalekamp MADH. Angiotensin production in the heart: a quantitative study with use of radiolabelled angiotensin infusions. Circulation 1998;98:73 – 81. [41] Sadoshima J, Xu Y, Slayter HS, Izumo S. Autocrine release of angiotensin II mediates stretch-induced hypertrophy of cardiac myocytes in vitro. Cell 1993;95:977 – 84. [42] Lindpaintner K, Jin M, Wilhelm MJ, Suzuki F, Linz W, Schelkensi BA, et al. Intracardiac generation of angiotensin and its physiological role. Circulation 1988;77(Suppl I):I-18 – 23. [43] Malhotra R, Sadoshima J, Broscius FC, Izumo S. Mechanical stretch and angiotensin II differentially upregulate the renin angiotensin system in cardiac myocytes in vitro. Circ Res 1999;85:137 – 46. [44] Lee YA, Liang CS, Lee HA, Lindpaintner K. Local stress, not systemic factors regulate gene expression of cardiac renin – angiotensin system in vivo: a comprehensive study of all its components in the dog. Proc Natl Acad Sci U S A 1996;93:11035 – 40. [45] De Mello WC, Crespo MJ. Correlation between changes in morphology, electrical properties and angiotensin converting enzyme activity in the failing heart. Eur J Pharmacol 1999;378:187 – 94. [46] Harada E, Yoshimura M, Yasue H, Nakagawa O, Nkagawa M, Harada M, et al. Aldosterone induces ACE gene expression in cultured neonatal rat cardiomyocytes. Ciculation 2001;104:137 – 9. [47] De Mello WC. Influence of intracellular renin on heart cell communication. Hypertension 1995;25:1172 – 7. [48] Sen I, Rajasekaran AK. Angiotensin II-binding protein in adult and neonatal rat heart. J Mol Cell Cardiol 1991;23:563 – 72. [49] Sugiura N, Hagiwara H, Hirose S. Molecular cloning of porcine soluble angiotensin binding protein. J Biol Chem 1992;267:18067 – 72. [50] Tang SS, Rogg H, Schumaker R, Dzau VJ. Characterization of nuclear angiotensin II-binding sites in rat liver and comparison with plasma membrane receptors. Endocrinology 1992;131:347 – 75. [51] Booz GW, Conrad KM, Hess AL, Singer HA, Baker KM. Angiotensin II-binding sites on hepatic nuclei. Endocrinology 1992;130:3641 – 9. [52] Re RN, Parab M. Effects of angiotensin II on RNA synthesis by isolated nuclei. Life Sci 1984;34:647 – 51. [53] Eggena P, Zhu JH, Sereevinyayut S, Girdani M, Clegg K, Andersen PC, et al. Hepatic angiotensin II nuclear receptors and transcription of growth-related factors. J Hypertens 1996;14:961 – 8. [54] Fu ML, Schulze W, Wallukat G, Elies R, Eftekhari P, Hjalmarson A, et al. Immunochemical localization of angiotensin II receptor (AT1) in the heart with anti-peptide antibody showing a positive chronotropic effect. Recept Channels 1998;6:99 – 111. [55] Kato A, Sugiura N, Hagiwara H, Hirose S. Cloning, amino acid sequence and tissue distribution of porcine thimet oligopeptidase. A comparison with soluble angiotensin-binding protein. Eur J Biochem 1994;221:159 – 65. [56] De Mello WC. Intracellular angiotensin II regulates the inward calcium current in cardiac myocytes. Hypertension 1998;32:976 – 82. [57] Mazzolai L, Nussberger J, Aubert JF, Brunner DB, Gabbiani G, Brunnet HR, et al. Blood pressure-independent cardiac hypertrophy induced by locally activated renin – angiotensin system. Hypertension 1998;31:1324 – 30. [58] Mazzolai L, Pedrazzini T, Nicoud F, Gabbiani G, Brunner HR, Nussberger J. Increased cardiac angiotensin II levels induce right and left ventricular hypertrophy in normotensive mice. Hypertension 2000;35: 985 – 91.
Invited review
Effect of extracellular and intracellular angiotensins on heart cell function; on the cardiac renin–angiotensin system Walmor De Mello * Department of Pharmacology, School of Medicine, Medical Sciences Campus, UPR, PO Box 365067, San Juan 00936-5067, PR, USA Received 12 September 2002; received in revised form 31 March 2003; accepted 5 April 2003
Abstract In this manuscript, I presented up-to-date evidence that intracellular and extracellular angiotensins have an important regulatory effect on the processes of heart cell communication and inward calcium current and that aldosterone modulates the effect of angiotensin II (Ang II) on the electrical properties of the heart. Moreover, I discussed the most relevant information about the origin of cardiac renin, the presence of a cardiac renin – angiotensin aldosterone system and its possible relevance for heart cell physiology and pathology. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Intracellular; Extracellular; Intracrine; Angiotensins; Renin; Aldosterone
The major function of cardiac muscle is contraction which propels the blood through systemic and pulmonary circulation. However, we must not forget that the heart is an outstanding electrochemical machine in which the energy stored by the membrane potential is periodically released, permitting the coupling between electrical and mechanical processes. Not only the ionic currents flowing through the surface cell membrane but the intercellular communication process mediated by gap junctions, are influenced by different factors including hormones. Indeed, evidence is available that angiotensin II (Ang II) regulates heart contractility, cell coupling, impulse propagation and is involved in the regulation of growth [1– 4]. These effects are mediated by Ang II receptors [5], which are 7-transmembrane domain receptors whose primary structures have been established by molecular cloning [6]. Exposure of AT1 receptors to Ang II is followed by translocation of the receptor to intracellular vesicles [7] and internalization of the Ang II AT1 receptor complex which occurs with a half-life of < 2 min [8,9]. The role of an internalized Ang II – receptor complex on heart cell function or as a source of intracellular Ang II merits further studies. The activation of AT2 receptors counteracts the AT1 receptor-induced progression of interstitial fibrosis and induces apoptosis [10] which is also induced by activation of AT1 receptors [11]. * Tel.: +1-787-766-4441; fax: +1-787-282-0568. E-mail address: [email protected] (W. De Mello).
It has been shown that Ang II added to the bath solution, reduces the gap junction conductance in cell pairs isolated from the ventricle of cardiomyopathic hamsters at an advanced stage of the disease [2,3]. This effect of Ang II, which appeared within seconds, was suppressed by losartan indicating that its effect was related to the activation of AT1 receptors. Moreover, the decline on cell coupling depends on the activation of protein kinase C [2,3]. Evidence is available that connexin 43, a major gap junction protein, is a MAP kinase substrate and that phosphorylation of Ser255, Ser279 and Ser282 initiates the downregulation of gap junction communication. Indeed, the levels of tyrosine phosphorylated connexin 43 are increased in the failing heart [12] supporting the idea that tyrosine phosphorylation is involved in the low values of junctional conductance found in the heart of cardiomyopathic hamsters [13]. Because it is known that tyrosine phosphorylation plays an important role on Ang II-mediated signal transduction [14], it is reasonable to think that it is also involved in the effect of the peptide on heart cell coupling. It has been found that AT1 receptor activation leads to an increased tyrosine phosphorylation of several intracellular substrates and that the effect of Ang II on remodeling is achieved through the activation of JAK –STAT pathways [15,16]. Moreover, c-Jun N-terminal kinase activation, a stress – activated protein kinase, mediates downregulation of connexin 43 in cardiomyocytes [17] but the relationship between this finding and the remodeling of gap junctions during pathological states needs to be clarified.
0167-0115/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0167-0115(03)00121-6
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W. De Mello / Regulatory Peptides 114 (2003) 87–90
The cell uncoupling elicited by Ang II seen in the ventricle of cardiomyopathic hamsters, at an advanced stage of heart failure, shows that the activation of the plasma renin – angiotensin system induces slow conduction and reentry—two major causes of cardiac arrhythmias [18]. Moreover, the local application of Ang II to different spots of the failing ventricle showed an increase in action potential duration in some cells but not in other [18]. This means that the peptide elicits an increased dispersion of the action potential duration (and consequently of QRS)—a cause of cardiac arrhythmias [18]. The mechanism by which Ang II increments the action potential duration is not known but there is the possibility that it is related to a change in potassium conductance (see Ref. [19]). It has been shown that Ang II is a cardiovascular risk factor [20] and that ACE inhibitors like enalapril, increase heart cell coupling and impulse propagation, an effect in part related to the increment of the Na – K pump and membrane hyperpolarization [21,22].
1. Aldosterone modulates the effect of Ang II It is known that Ang II releases aldosterone from the adrenal gland. Therefore, it is important to know if aldosterone alters the effect of Ang II on heart muscle. Recently, it was found that the effect of Ang II on the electrical properties of adult rat heart is modulated by aldosterone [23]. Indeed, in rats treated with aldosterone for 48 h, Ang II, added to the extracellular medium, increases the action potential duration and refractoriness contrary to its effect in the controls in which both parameters are reduced by the same dose of Ang II [23]. This means that the arrhythmogenic action of Ang II in rat heart is reversed by aldosterone. Because the increment in action potential duration elicited by Ang II in aldosterone treated rats was suppressed by verapamil it is reasonable to conclude that it is related to an increased inward calcium current. Interestingly, the effect of isoproterenol on cardiac excitability and action potential duration was not altered by aldosterone [23]. These findings show, for the first time, that the effect of Ang II on the electrical and probably in other functions of the heart must be evaluated considering the presence or absence of aldosterone. Recently, it has been shown that aldosterone receptors are present in the heart [24] and that aldosterone is produced in the heart [25]. Chronic treatment with Ang II as well as a low sodium/high potassium diet, increased production of aldosterone [26,27] what raises the possibility that there is a cross-talk between angiotensin and aldosterone in the heart. Further studies are needed to confirm this hypothesis.
2. On the hypothesis of cardiac renin– angiotensin system There is evidence that there is a local renin –angiotensin system (RAS) in the heart, an idea based on the
demonstration that elements of the RAS cascade (renin, angiotensinogen, Ang I, Ang II and angiotensin converting enzyme (ACE)) are present in this tissue [28,29]. In normal heart, however, renin mRNA levels are extremely low or undetectable in nephrectomized animals [30] what signifies that the uptake of renin from plasma is the major source of cardiac renin [31]. The possibility exists that renin is internalized by mannose-6 phosphate receptors for which glycosylation of renin is needed [32] or that renin is bound to an unidentified protein [33,34] and that Ang II is synthesized inside the cells. The contribution of plasma renin to cardiac renin was tested using transgenic mice expressing human renin in the liver (TTRhRen-A3) which were mated to mice expressing human angiotensinogen exclusively in the heart (MHChAgt-2). These results indicated low or undetectable angiotensin peptide in the heart of single transgenic animals while double transgenic mice showed a remarkable increase of cardiac levels of Ang I and Ang II, indicating that plasma renin is able to act on its substract within the heart [35]. Recently, it has been shown that prorenin is internalized into cardiomyocytes independently of glycosylation and that this process is of functional significance because it leads to generation of angiotensins [36]. Other studies, however, indicate that prorenin induces myocyte proliferation, an effect unrelated to intracellular Ang II [37]. An important finding was that the heart expresses an alternative renin transcript (exon 1A renin) which is maintained intracellularly [38]. This expression, which is markedly enhanced after myocardial ischemia [38], supports the notion that there is an intracellular renin – angiotensin system [39]. Previous observations [40] showed that > 90% of cardiac Ang I and more than 75% of cardiac Ang II are synthesized at cardiac sites. Indeed, experimental evidence indicates that angiotensins are generated locally and released by the heart [41,42]. An important question is which factor or factors lead to activation of the cardiac renin – angiotensin system. Studies of Malhotra et al. [43] indicated that mechanical stretch increases the expression of renin and angiotensinogen genes in neonatal cardiac myocytes. Local mechanical stress rather then systemic factors seems to regulate the expression of these genes [44] because right ventricular hypertrophy, for instance, increases the mRNA levels of ACE, AT2 receptors and decreases the expression of AT1 receptors in the right ventricle but no change in expression of renin and angiotensinogen genes were found in the left ventricle [44]. In the failing heart of cardiomyopathic hamsters, the ACE activity is not increased at 2 months of age but it is appreciably enhanced afterwards when overt heart failure is seen [45]. Because aldosterone induces angiotensin converting enzyme gene expression in cultured cardiac cells [46], the question remains if aldosterone in involved in the activation of the cardiac renin – angiotensin system.
W. De Mello / Regulatory Peptides 114 (2003) 87–90
Previous studies from our laboratory indicated that intracellular renin caused a decrease of gap junction communication in heart muscle [47]. Furthermore, intracellular dialysis of Ang I into myocytes of cardiomyopathic hamsters, at an advanced stage of the disease, elicited total suppression of cell communication, an effect related to its conversion to Ang II because the effect of Ang I was greatly reduced by intracellular enalaprilat [2]. As a matter of fact, intracellular dialysis of Ang II into these cells abolished cell coupling, an effect suppressed by intracellular but not by extracellular losartan. These observations might indicate that the effect of intracellular Ang II is dependent upon the presence of an intracellular receptor similar to AT1. Evidence is available that there is a soluble angiotensinbinding receptor cytosolic protein with a molecular weight of 75 kDa [48 – 50]. Moreover, nuclear and chromatin Ang II receptors have been identified [51 – 53]. Immunochemical studies also indicated the presence of intracellular Ang II receptors in cardiomyocytes and fibroblasts [54]. This finding contrasts with previous studies of Kato et al. [55], which demonstrated that the porcine soluble angiotensinbinding protein is a microsomal endopeptidase—a finding that does not support the idea of an intracellular Ang II receptor. Intracellular injection of Ang II into ventricular cells of intact ventricle of cardiomyopathic hamsters, at 4 months of age, demonstrated an increase in action potential duration and refractoriness but cardiac arrhythmias appeared when larger amounts of Ang II (10 9 M) were injected (De Mello, in preparation). These changes elicited by intracellular Ang II, were suppressed by intracellular losartan (De Mello, in preparation). Additional experiments demonstrated that intracellular dialysis of Ang II reduces the inward calcium current in isolated cardiomyocytes of adult rats but increases it in myocytes of Golden hamsters [56], which suggests that intracellular Ang II is involved in the regulation of heart contractility, an effect that varies with the species. The increase of calcium current, which was related to the activation of protein kinase C [56], might be important for the failing heart. Indeed, Ang II dialyzed into ventriclar cells of cardiomyopathic hamsters cells, elicted an increment of the inward calcium current. Because the effect of intracellular Ang II on inward calcium current was not suppressed by losartan applied to the extracellular fluid, it is reasonable to think that the peptide is acting intracellularly (De Mello, unpublished). Transgenic animal models have been used to investigate the role of cardiac renin – angiotensin system. Overexpression of angiotensinogen gene in normal heart muscle cells caused an increment of the angiotensin II concentration in right and left ventricle and elicited hypertrophy of both ventricles without any change in arterial blood pressure [57,58]. These observations indicate that cardiac Ang II is able to cause ventricular hypertrophy independently of hemodynamic alterations.
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3. Conclusions The evidence gathered thus far supports the view that there is a cardiac renin – angiotensin system which is activated during pathological states such as heart failure. However, the debate continues about the site of Ang II synthesis. Extracellular and intracellular angiotensins play an important role on the control of heart cell function and aldosterone modulates the effect of Ang II on electrical properties of the heart. The role of intracellular angiotensins on cardiac cell function might indicate that therapeutic procedures must be developed to control intracellular angiotensin and renin levels. Further studies on the role of intracellular Ang II and aldosterone on cardiac functions will provide penetrating insights into the intricacies of the interactions between Ang II and aldosterone and their implications to heart cell biology.
Acknowledgements This work was supported by grants from the American Heart Association and NIH (HL-34148, 532943).
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