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Fibrosis rather than blood pressure determines cardiac BNP expression in mice
Regulatory Peptides 116 (2003) 95 – 100 www.elsevier.com/locate/regpep
Fibrosis rather than blood pressure determines cardiac BNP expression in mice Thomas Walther a,*,1, Katrin Klostermann b, Silvia Heringer-Walther a, Heinz-Peter Schultheiss a, Carsten Tscho¨pe a, Holger Stepan b a
Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany b Department of Obstetrics and Gynecology, University of Leipzig, Leipzig, Germany Received 7 January 2003; received in revised form 30 June 2003; accepted 28 July 2003
Abstract Background: Since first reports demonstrated interactions between the natriuretic peptide (NPS) and renin – angiotensin system (RAS), our experiments should clarify whether cardiac brain natriuretic peptide (BNP) is regulated in mice genetically altered for components of the RAS. Methods and results: The study was carried out in hypotensive AT1- and angiotensinogen (ANG)-, and normotensive AT2-knockout mice, and in hypertensive animals overexpressing ANG and wildtype controls of each genotype. Ventricular BNP expression was analyzed by RNase-protection assay (RPA) (n = 6). Cardiac fibrosis was visualized by Sirius red staining. While ANG overexpression increases cardiac BNP-mRNA expression (1035 F 210 vs. wildtype: 405 F 95 in PSL/mm2, P < 0.01), its deficiency had no influence. Both AT1- and AT2knockouts showed significantly decreased BNP-mRNA concentrations (AT1: 21 F 6 vs. wildtype: 139 F 28 in PSL/mm2, P < 0.001; AT2: 8 F 2 vs. 19 F 3 in PSL/mm2, P < 0.05). These alterations correlate to reduced cardiac fibrosis in AT2-deficient animals, and an unchanged matrix content in ANG knockouts. Conclusions: Increased BNP-mRNA levels in hypertensive ANG-overexpressing mice and decreased BNP in hypotensive AT1-deficient animals suggest that this mRNA expression is blood pressure-dependent. However, the observed alterations of fibrosis and the unchanged BNP in hypotensive ANG knockouts and impaired BNP-mRNA expression in normotensive AT2deficient mice demonstrate a direct interaction of the RAS and NPS that is fibrosis- rather than blood pressure-dependent. D 2003 Elsevier B.V. All rights reserved. Keywords: Angiotensin; Blood pressure; Fibrosis; Gene expression; Natriuretic peptide
1. Introduction The role of endogenous vasoactive peptides as regulators of cardiac function in health and disease is an emerging area of cardiovascular research. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are secreted in an endocrine fashion mainly from the heart. Both peptides serve to maintain natriuresis, inhibit aldosterone secretion [1], and reduce intravascular volume and pressure. Their combined actions contribute to arterial pressure and cardiac filling pressure and output [2]. ANP and BNP have become predictive for left ventricular hypertrophy and dysfunction * Corresponding author. Tel.: +49-30-8445-4258; fax: +49-30-84454648. E-mail address: [email protected] (T. Walther). 1 Present address: Department of Pharmacology, Erasmus MC Dr. Molewaterplein 50, Rotterdam, The Netherlands. 0167-0115/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2003.07.003
[3]. However, they respond differently to cardiac short- and long-term volume load in the circulation [4], acute hyperinsulinemia [5], and cardiac hypertrophy compensatory to hypertension [6]. Recently, the importance of BNP in clinical diagnostics of cardiovascular diseases has increased, since this hormone has gathered prognostic value in patients with acute coronary syndromes [7] and BNP-guided treatment of heart failure reduced cardiovascular morbidity [8]. Interactions between ANP and the renin – angiotensin system (RAS) have been demonstrated previously. Angiotensin converting enzyme (ACE) inhibition and angiotensin II type 1 receptor antagonism have been reported to influence the renal sodium and water handling and albuminuria during infusion of ANP into healthy volunteers [9]. It has also been shown that synthesis and secretion of ANP and BNP differ in hypertensive TGR(mREN-2)27 transgenic rats that overexpress the murine Ren-2d renin gene. Whereas ANP mRNA and circulating peptide concentrations are
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increased, BNP plasma levels seem to be unaffected by overexpression of renin [10]. Although there are only a few studies indicating a direct link between BNP and RAS [11], Murdoch et al. [12], for example, have demonstrated that the titration of ACE inhibitor therapy according to plasma BNP is associated with a stronger inhibition of the RAS and a significant decrease of heart rate compared with conventional therapy. Expression of cardiac BNP is triggered, for instance, by endothelin [13] or interleukin-1h [14]. Since both peptides are stimulated by angiotensin II (Ang II), it could be expected that alterations of the RAS influence BNP expression in the heart. Therefore, the aim of our study was to investigate alterations of BNP expression in mice deficient for the angiotensin receptors AT1A [15] or AT2 [16] and in those deficient for [17] or over-expressing [18] angiotensinogen (ANG). The experiments should further distinguish between direct effects of circulating or cardiac Ang II or blood pressure-mediated changes of BNP expression, respectively.
sequencing and sequence alignments (program BLAST; National Center for Biotechnology Information). T7 polymerase transcribed a 290-bp-long radioactive probe complementary to 244 nucleotides of the BNP mRNA. A total of 653 bp from rat collagen I cDNA were protected, as well as 127 or 130 bp from control rL32 or GAPDH sequences, respectively. Identification of mRNA specific for mouse BNP or collagen I was effected by RNaseprotection assay (RPA), using the Ambion RPA II kit (AMS Biotechnology, Witney, UK) as described previously [20]. Five micrograms of total RNA per sample were hybridized with approximately 50,000 cpm for BNP or collagen I and 30,000 cpm for GAPDH or rL32 of the radiolabelled antisense probes in the same assay. The hybridized fragments protected from RNase A + T1 digestion were separated by electrophoresis on a denaturing gel (5% polyacrylamide, 8 M urea) and analyzed using a FUJIX BAS 2000 Phospho-Imager system (Raytest, Straubenhardt, Germany). Quantitative analysis was performed by measuring the intensity of the BNP or collagen I bands normalized by the intensity of the GAPDH or rL32 bands.
2. Materials and methods
2.3. Sirius red staining, immunohistology and quantification of collagen
2.1. Animals This research was in compliance with the Guide for the Care and Use of Laboratory Animals published by the Office for Protection against Research Risks (OPRR) of the US National Institutes of Health, Washington, DC (NIH Publication No. 85-23, revised 1996) and the institutional Animal Ethics Committee. Male mice of different transgenic strains (NMRI123 own breeding colony; breeding couples were provided by Dr. Coffman [AT1A], Dr. Inagami [AT2], and Dr. Murakami [TLM]) were kept under standardized conditions with an artificial 12-h dark –light cycle, with access to food and water ad libitum. Three- to four-month-old mice were killed by cervical dislocation. All ventricular cardiac tissue samples were collected at the same time each day and immediately snap-frozen in liquid nitrogen (n = 6 tissue samples per genotype for each experiment). 2.2. RNase-protection assay Total RNA was isolated from tissues by using the TRIzolk reagent (Life Technologies, Eggenstein, Germany) followed by chloroform-isopropanol extraction, according to the protocol of the manufacturer. Via polymerase chain reaction a fragment with a length of 244 bp was amplified from mouse BNP-cDNA using the 5Vprimer: GCA TGG ATC TCC TGA AGG and the 3Vprimer: GCG CTG CCT TGA GAC CGA AGG and subcloned in a Tvector (Promega, Mannheim, Germany). The primers were designed using the mouse genomic sequence of the BNP gene [19]. The inserted fragments were confirmed by
Serial 5 mm thick, transverse cryosections of Tissue TecR embedded hearts were placed on Poly-L-Lysine precoated slides and fixed in cold acetone for 10 min. The collagen content of the picrosirius red (Polyscience, USA) stained sections was measured under circularly polarized light according to previously published methods [21]. 2.4. Statistical analysis Results are expressed as mean F S.D. Statistical comparisons were made by one-way analysis of the variance and Tukey HSD test. Statistical significance was considered as P < 0.05.
3. Results BNP mRNA could be detected in all cardiac tissue samples. Hypertensive male mice overexpressing angiotensinogen (NMRI123) with elevated Ang II levels [18] showed significantly increased cardiac BNP-mRNA concentrations (overexpression: 1035 F 210 vs. wildtype: 405 F 95 in PSL/mm2, P < 0.01; Fig. 1). To identify the receptor responsible for the Ang II-mediated control of BNP expression, animals deficient for the AT1A or AT2 receptor were analyzed. Surprisingly, the hypotensive AT1-knockout mice as well as the normotensive AT2-knockout mice showed a significant decrease of BNP-mRNA concentrations. Whereas the AT1-knockout animals show only 15% of the BNP
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Fig. 1. Quantification of cardiac BNP-mRNA expression in mice overexpressing angiotensinogen (NMRI123 +) vs. wildtype (NMRI123 ) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/ mm2 after normalization to GAPDH-mRNA levels (n = 6 per group); *P < 0.01.
Fig. 3. Quantification of cardiac BNP-mRNA expression in mice lacking the AT2 receptor (AT2 / ) vs. wildtype (AT2 +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/mm2 after normalization to GAPDH-mRNA levels (n = 6 per group); *P < 0.05.
Fig. 2. Quantification of cardiac BNP-mRNA expression in mice lacking the AT1 receptor (AT1 / ) vs. wildtype (AT1 +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/mm2 after normalization to GAPDH-mRNA levels (n = 6 per group); *P < 0.0005.
Fig. 4. Quantification of cardiac BNP-mRNA expression in mice lacking angiotensinogen (TLM / ) vs. wildtype (TLM +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/mm2 after normalization to GAPDH-mRNA levels (n = 6 per group).
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expression in comparison to the controls, cardiac BNP mRNA of the AT2 knockout animals is half of the control animals (AT1 knockout: 21 F 6 vs. wildtype: 139 F 28 in PSL/mm2, P < 0.0005; AT2 knockout: 8 F 2 vs. 19 F 3, P < 0.05; Figs. 2 and 3). However, hypotensive animals with disrupted angiotensinogen gene (TLM) do not show significantly altered
cardiac BNP mRNA (TLM knockout: 86 F 17 vs. wildtype: 65 F 13 in PSL/mm2; Fig. 4). The quantification of Sirius red staining demonstrated that fibrosis was significantly reduced by 30% in AT2-deficient mice but unchanged in TLM mice (Fig. 5). Alterations in matrix composition have been further determined by a collagen I-specific RNase-protec-
Fig. 5. Leftventricular extracellular matrix content demonstrated by (A) Sirius red staining in left ventricular sections of mice lacking angiotensinogen (TLM / ) and wildtype (TLM +/+) animals and lacking the angiotensin AT2 receptor (AT2 / ) vs. their wildtype (AT2 +/+). (B) Quantification of Sirius red staining in all four strains summarizing data from 30 counts per one cryosection in six animals per strain.
T. Walther et al. / Regulatory Peptides 116 (2003) 95–100
Fig. 6. Quantification of cardiac collagen I (Col I)-mRNA expression in mice lacking the AT2 receptor (AT2 / ) vs. wildtype (AT2 +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/ mm2 after normalization to rL32-mRNA levels (n = 6 per group); *P < 0.05.
tion assay for AT2-deficient mice (Fig. 6). As indicated by Sirius red staining on protein level, the collagen ImRNA concentrations are significantly lower in these AT2 knockouts compared to their wildtype controls ( P < 0.05).
4. Discussion This is the first description of cardiac BNP expression in mice genetically altered for components of the RAS. Taking the results from angiotensinogen-overexpression and AT1Adeficient animals, our data would clearly indicate the linearity between the level of Ang II-mediated stimulation of the AT1 receptor and cardiac BNP expression. Nevertheless, these data could not distinguish between direct peptide effects or secondary effects mediated by blood pressure alterations. Since we demonstrate that normotensive mice lacking the AT2 receptor [16,22,23] show also an impaired cardiac BNP expression, we can exclude an exclusive influence of the blood pressure and have to assume a direct effect of the cardiac RAS on BNP expression. The existence of a blood pressure-independent factor is also indicated by our observation that hypotensive angiotensinogen-deficient animals show unchanged cardiac BNP-mRNA levels. There seems to be a discrepancy between the results of both
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hypotensive animal models (angiotensinogen- or AT1-deficient mice), since in both models the Ang II/AT1-axis is disrupted which should lead to the same regulation of BNP. We have to consider that the lack of the AT1 receptor could cause an additional stimulation of the AT2 receptor and an increase in other cardiovascular active Ang metabolites, whereas the angiotensinogen-deficient animals also lack the effects of AT2 stimulation such as Ang IV and Ang(1-7) signaling. This is of particular interest, since Ang-(1-7) has been characterized as a cardioprotective factor [24]. Additionally, the difference could be also mediated by a blocked Ang II-AT1B axis in angiotensinogen-deficient animals, whereas the AT1B is still present in AT1A knockouts. If elevated BNP is a marker for cardiac failure [8,12] and the angiotensinogen-deficient animals have higher BNP-mRNA concentrations compared to AT1 knockouts, we have to conclude that the selective deletion of the ‘‘bad’’ angiotensin receptor AT1 is cardioprotective compared to the disruption of the whole RAS in angiotensinogen-deficient mice where also positive components like Ang-(1-7) are switched off. Secondly, Sakata et al. [6] conclude also in their paper that the BNP increase is directly controlled by the AT1 receptor. This would confirm our data of dramatic reduction in BNP expression in animals lacking this receptor. Therefore, further cell culture experiments have to clarify, whether the treatment of cardiomyocytes with Ang II would lead to a direct up-regulation of BNP mRNA or whether the observed reduction of BNP after AT1 blockade is mediated indirectly by an improved cardiac function. What other factors than blood pressure may trigger cardiac BNP-mRNA expression? Firstly, cardiac fibrosis is associated with elevated BNP concentrations in the heart [25]. Mice deficient for the BNP gene (NppB knockout) indicate that this elevation is not just a marker of fibrotic status. These animals are characterized by normotension but a rise in cardiac fibrosis which indicates that BNP acts locally as an antifibrotic factor in the heart [26,27] concluding that high BNP in the state of fibrosis is a cardiac rescue mechanism to prevent further deterioration. Our group recently demonstrated that increased fibrosis in angiotensinogen-overexpressing mice correlated with a rise in BNP mRNA [28]. Since our present data clearly demonstrate that reduced fibrosis correlates to a lower BNP-mRNA concentration in normotensive AT2-deficient animals and that there is no change in fibrosis or BNP mRNA in hypotensive angiotensinogen-deficient mice, it is fibrosis rather than blood pressure that regulates cardiac BNP expression. This postulate is also supported by findings of Sakata et al. [6] demonstrating that BNP-gene expression is not enhanced by initial compensatory hypertrophy but is enhanced by left ventricular fibrosis. Following this argumentation, the reduction of BNP mRNA in normotensive AT2-knockout animals implies a profibrotic function of this receptor that is blocked by AT2 deficiency. This is supported by the observation of Ichihara et al. [29] who demonstrated that animals
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with the genetic deletion of the AT2 receptor do not develop cardiac fibrosis in Ang II-induced chronic hypertension. In summary, the observed alterations in fibrosis and the unchanged BNP in hypotensive angiotensinogen knockouts and impaired BNP-mRNA expression in normotensive AT2-deficient mice demonstrate a direct influence of components of the RAS on the expression of BNP. This interaction is mediated more by fibrosis than blood pressure changes.
Acknowledgements The study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG; WA-1441/1). We thank the Humboldt Foundation for the post-doctoral fellowship to S. H-W.
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[12] Murdoch DR, McDonagh TA, Byrne J, Blue L, Farmer R, Morton JJ, et al. Titration of vasodilator therapy in chronic heart failure according to plasma brain natriuretic plasma concentration: randomized comparison if the hemodynamic and neuroendocrine effects of tailored versus empirical therapy. Am Heart J 1999;138:1005 – 6. [13] Bruneau BG, Piazza LA, de Bold AJ. BNP gene expression is specifically modulated by stretch and ET-1 in a new model of isolated rat atria. Am J Physiol 1997;273:2678 – 86. [14] He Q, LaPointe MC. Interleukin-1beta regulates the human brain natriuretic peptide promoter via Ca(2+)-dependent protein kinase pathways. Hypertension 2000;35:292 – 6. [15] Ito M, Oliveirio MI, Mannon PJ, Best CF, Maeda N, Smithies O, et al. Regulation of blood pressure by the type 1A angiotensin II receptor gene. PNAS 1995;92:3521 – 5. [16] Ichiki T, Labosky PA, Shiota C, Okuyama S, Imagawa Y, Fogo A, et al. Effects on blood pressure and exploratory behavior of mice lacking angiotensin II type-2 receptors. Nature 1995;377:748 – 50. [17] Tanimoto K, Sugiyama F, Goto Y, Ishida J, Takimoto E, Yagami K, et al. Angiotensinogen-deficient mice with hypotension. J Biol Chem 1994;269:31334 – 7. [18] Kimura J, Mullins JJ, Bunnemann B, Metzger R, Hilgenfeldt U, Zimmermann F, et al. High blood pressure in transgenic mice carrying the rat angiotensinogen gene. EMBO J 1992;11:821 – 7. [19] Ogawa Y, Itoh H, Tamura N, Suga S, Yoshimasa T, Uehira M, et al. Molecular cloning of the complementary DNA and gene that encode mouse brain natriuretic peptide and generation of transgenic mice that overexpress the brain natriuretic peptide gene. J Clin Invest 1994;93: 1911 – 21. [20] Stepan H, Leitner E, Walter K, Bader M, Schultheiss HP, Faber R, et al. Gestational regulation of the gene expression of C-type natriuretic peptide in mouse reproductive and embryonic tissue. Regul Pept 2001;102:9 – 13. [21] Pauschinger M, Knopf D, Petschauer S, Doerner A, Poller W, Schwimmbeck PL, et al. Dilated cardiomyopathy is associated with significant changes in collagen type I/III ratio. Circulation 1999;99: 2750 – 6. [22] Hein L, Barsh GS, Pratt RE, Dzau VJ, Kobilka BK. Behavioural and cardiovascular effects of disrupting the angiotensin II type-2 receptor in mice [published erratum appears in Nature 1996 Mar 28;380(6572):366]. Nature 1995;377:744 – 7. [23] Gross V, Walther T, Milia AF, Walter K, Schneider W, Luft FC. Left ventricular function in mice lacking the AT2 receptor. J Hypertens 2001;19:967 – 76. [24] Ferreira AJ, Santos RA, Almeida AP. Angiotensin-(1-7): cardioprotective effect in myocardial ischemia/reperfusion. Hypertension 2001; 38:665 – 8. [25] Wei CM, Heublein DM, Perella MA, et al. Natriuretic peptide system in human heart failure. Circulation 1993;88:1004 – 9. [26] Tamura N, Ogawa Y, Chusho H, Nakamura K, Nakao K, Suda M, et al. Cardiac fibrosis in mice lacking brain natriuretic peptide. PNAS 2000;97:4239 – 44. [27] Ogawa Y, Tamura N, Chusho H, Nakao K. Brain natriuretic peptide appears to act locally as an antifibrotic factor in the heart. Can J Physiol Pharmacol 2001;79:723 – 9. [28] Kang NL, Walther T, Tian XL, Bohlender J, Fukamizu A, Ganten D, et al. Reduced hypertension-induced end-organ damage in mice lacking cardiac and renal angiotensinogen synthesis. J Mol Med 2002;80: 359 – 66. [29] Ichihara S, Senbonmatsu T, Price E, Ichiki T, Gaffney A. Inagami. Angiotensin II Type 2 receptor is essential for left ventricular hypertrophy and cardiac fibrosis in chronic angiotensin II-induced hypertension. Circulation 2001;104:346 – 51.
Fibrosis rather than blood pressure determines cardiac BNP expression in mice Thomas Walther a,*,1, Katrin Klostermann b, Silvia Heringer-Walther a, Heinz-Peter Schultheiss a, Carsten Tscho¨pe a, Holger Stepan b a
Department of Cardiology and Pneumology, University Hospital Benjamin Franklin, Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany b Department of Obstetrics and Gynecology, University of Leipzig, Leipzig, Germany Received 7 January 2003; received in revised form 30 June 2003; accepted 28 July 2003
Abstract Background: Since first reports demonstrated interactions between the natriuretic peptide (NPS) and renin – angiotensin system (RAS), our experiments should clarify whether cardiac brain natriuretic peptide (BNP) is regulated in mice genetically altered for components of the RAS. Methods and results: The study was carried out in hypotensive AT1- and angiotensinogen (ANG)-, and normotensive AT2-knockout mice, and in hypertensive animals overexpressing ANG and wildtype controls of each genotype. Ventricular BNP expression was analyzed by RNase-protection assay (RPA) (n = 6). Cardiac fibrosis was visualized by Sirius red staining. While ANG overexpression increases cardiac BNP-mRNA expression (1035 F 210 vs. wildtype: 405 F 95 in PSL/mm2, P < 0.01), its deficiency had no influence. Both AT1- and AT2knockouts showed significantly decreased BNP-mRNA concentrations (AT1: 21 F 6 vs. wildtype: 139 F 28 in PSL/mm2, P < 0.001; AT2: 8 F 2 vs. 19 F 3 in PSL/mm2, P < 0.05). These alterations correlate to reduced cardiac fibrosis in AT2-deficient animals, and an unchanged matrix content in ANG knockouts. Conclusions: Increased BNP-mRNA levels in hypertensive ANG-overexpressing mice and decreased BNP in hypotensive AT1-deficient animals suggest that this mRNA expression is blood pressure-dependent. However, the observed alterations of fibrosis and the unchanged BNP in hypotensive ANG knockouts and impaired BNP-mRNA expression in normotensive AT2deficient mice demonstrate a direct interaction of the RAS and NPS that is fibrosis- rather than blood pressure-dependent. D 2003 Elsevier B.V. All rights reserved. Keywords: Angiotensin; Blood pressure; Fibrosis; Gene expression; Natriuretic peptide
1. Introduction The role of endogenous vasoactive peptides as regulators of cardiac function in health and disease is an emerging area of cardiovascular research. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are secreted in an endocrine fashion mainly from the heart. Both peptides serve to maintain natriuresis, inhibit aldosterone secretion [1], and reduce intravascular volume and pressure. Their combined actions contribute to arterial pressure and cardiac filling pressure and output [2]. ANP and BNP have become predictive for left ventricular hypertrophy and dysfunction * Corresponding author. Tel.: +49-30-8445-4258; fax: +49-30-84454648. E-mail address: [email protected] (T. Walther). 1 Present address: Department of Pharmacology, Erasmus MC Dr. Molewaterplein 50, Rotterdam, The Netherlands. 0167-0115/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2003.07.003
[3]. However, they respond differently to cardiac short- and long-term volume load in the circulation [4], acute hyperinsulinemia [5], and cardiac hypertrophy compensatory to hypertension [6]. Recently, the importance of BNP in clinical diagnostics of cardiovascular diseases has increased, since this hormone has gathered prognostic value in patients with acute coronary syndromes [7] and BNP-guided treatment of heart failure reduced cardiovascular morbidity [8]. Interactions between ANP and the renin – angiotensin system (RAS) have been demonstrated previously. Angiotensin converting enzyme (ACE) inhibition and angiotensin II type 1 receptor antagonism have been reported to influence the renal sodium and water handling and albuminuria during infusion of ANP into healthy volunteers [9]. It has also been shown that synthesis and secretion of ANP and BNP differ in hypertensive TGR(mREN-2)27 transgenic rats that overexpress the murine Ren-2d renin gene. Whereas ANP mRNA and circulating peptide concentrations are
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increased, BNP plasma levels seem to be unaffected by overexpression of renin [10]. Although there are only a few studies indicating a direct link between BNP and RAS [11], Murdoch et al. [12], for example, have demonstrated that the titration of ACE inhibitor therapy according to plasma BNP is associated with a stronger inhibition of the RAS and a significant decrease of heart rate compared with conventional therapy. Expression of cardiac BNP is triggered, for instance, by endothelin [13] or interleukin-1h [14]. Since both peptides are stimulated by angiotensin II (Ang II), it could be expected that alterations of the RAS influence BNP expression in the heart. Therefore, the aim of our study was to investigate alterations of BNP expression in mice deficient for the angiotensin receptors AT1A [15] or AT2 [16] and in those deficient for [17] or over-expressing [18] angiotensinogen (ANG). The experiments should further distinguish between direct effects of circulating or cardiac Ang II or blood pressure-mediated changes of BNP expression, respectively.
sequencing and sequence alignments (program BLAST; National Center for Biotechnology Information). T7 polymerase transcribed a 290-bp-long radioactive probe complementary to 244 nucleotides of the BNP mRNA. A total of 653 bp from rat collagen I cDNA were protected, as well as 127 or 130 bp from control rL32 or GAPDH sequences, respectively. Identification of mRNA specific for mouse BNP or collagen I was effected by RNaseprotection assay (RPA), using the Ambion RPA II kit (AMS Biotechnology, Witney, UK) as described previously [20]. Five micrograms of total RNA per sample were hybridized with approximately 50,000 cpm for BNP or collagen I and 30,000 cpm for GAPDH or rL32 of the radiolabelled antisense probes in the same assay. The hybridized fragments protected from RNase A + T1 digestion were separated by electrophoresis on a denaturing gel (5% polyacrylamide, 8 M urea) and analyzed using a FUJIX BAS 2000 Phospho-Imager system (Raytest, Straubenhardt, Germany). Quantitative analysis was performed by measuring the intensity of the BNP or collagen I bands normalized by the intensity of the GAPDH or rL32 bands.
2. Materials and methods
2.3. Sirius red staining, immunohistology and quantification of collagen
2.1. Animals This research was in compliance with the Guide for the Care and Use of Laboratory Animals published by the Office for Protection against Research Risks (OPRR) of the US National Institutes of Health, Washington, DC (NIH Publication No. 85-23, revised 1996) and the institutional Animal Ethics Committee. Male mice of different transgenic strains (NMRI123 own breeding colony; breeding couples were provided by Dr. Coffman [AT1A], Dr. Inagami [AT2], and Dr. Murakami [TLM]) were kept under standardized conditions with an artificial 12-h dark –light cycle, with access to food and water ad libitum. Three- to four-month-old mice were killed by cervical dislocation. All ventricular cardiac tissue samples were collected at the same time each day and immediately snap-frozen in liquid nitrogen (n = 6 tissue samples per genotype for each experiment). 2.2. RNase-protection assay Total RNA was isolated from tissues by using the TRIzolk reagent (Life Technologies, Eggenstein, Germany) followed by chloroform-isopropanol extraction, according to the protocol of the manufacturer. Via polymerase chain reaction a fragment with a length of 244 bp was amplified from mouse BNP-cDNA using the 5Vprimer: GCA TGG ATC TCC TGA AGG and the 3Vprimer: GCG CTG CCT TGA GAC CGA AGG and subcloned in a Tvector (Promega, Mannheim, Germany). The primers were designed using the mouse genomic sequence of the BNP gene [19]. The inserted fragments were confirmed by
Serial 5 mm thick, transverse cryosections of Tissue TecR embedded hearts were placed on Poly-L-Lysine precoated slides and fixed in cold acetone for 10 min. The collagen content of the picrosirius red (Polyscience, USA) stained sections was measured under circularly polarized light according to previously published methods [21]. 2.4. Statistical analysis Results are expressed as mean F S.D. Statistical comparisons were made by one-way analysis of the variance and Tukey HSD test. Statistical significance was considered as P < 0.05.
3. Results BNP mRNA could be detected in all cardiac tissue samples. Hypertensive male mice overexpressing angiotensinogen (NMRI123) with elevated Ang II levels [18] showed significantly increased cardiac BNP-mRNA concentrations (overexpression: 1035 F 210 vs. wildtype: 405 F 95 in PSL/mm2, P < 0.01; Fig. 1). To identify the receptor responsible for the Ang II-mediated control of BNP expression, animals deficient for the AT1A or AT2 receptor were analyzed. Surprisingly, the hypotensive AT1-knockout mice as well as the normotensive AT2-knockout mice showed a significant decrease of BNP-mRNA concentrations. Whereas the AT1-knockout animals show only 15% of the BNP
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Fig. 1. Quantification of cardiac BNP-mRNA expression in mice overexpressing angiotensinogen (NMRI123 +) vs. wildtype (NMRI123 ) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/ mm2 after normalization to GAPDH-mRNA levels (n = 6 per group); *P < 0.01.
Fig. 3. Quantification of cardiac BNP-mRNA expression in mice lacking the AT2 receptor (AT2 / ) vs. wildtype (AT2 +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/mm2 after normalization to GAPDH-mRNA levels (n = 6 per group); *P < 0.05.
Fig. 2. Quantification of cardiac BNP-mRNA expression in mice lacking the AT1 receptor (AT1 / ) vs. wildtype (AT1 +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/mm2 after normalization to GAPDH-mRNA levels (n = 6 per group); *P < 0.0005.
Fig. 4. Quantification of cardiac BNP-mRNA expression in mice lacking angiotensinogen (TLM / ) vs. wildtype (TLM +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/mm2 after normalization to GAPDH-mRNA levels (n = 6 per group).
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expression in comparison to the controls, cardiac BNP mRNA of the AT2 knockout animals is half of the control animals (AT1 knockout: 21 F 6 vs. wildtype: 139 F 28 in PSL/mm2, P < 0.0005; AT2 knockout: 8 F 2 vs. 19 F 3, P < 0.05; Figs. 2 and 3). However, hypotensive animals with disrupted angiotensinogen gene (TLM) do not show significantly altered
cardiac BNP mRNA (TLM knockout: 86 F 17 vs. wildtype: 65 F 13 in PSL/mm2; Fig. 4). The quantification of Sirius red staining demonstrated that fibrosis was significantly reduced by 30% in AT2-deficient mice but unchanged in TLM mice (Fig. 5). Alterations in matrix composition have been further determined by a collagen I-specific RNase-protec-
Fig. 5. Leftventricular extracellular matrix content demonstrated by (A) Sirius red staining in left ventricular sections of mice lacking angiotensinogen (TLM / ) and wildtype (TLM +/+) animals and lacking the angiotensin AT2 receptor (AT2 / ) vs. their wildtype (AT2 +/+). (B) Quantification of Sirius red staining in all four strains summarizing data from 30 counts per one cryosection in six animals per strain.
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Fig. 6. Quantification of cardiac collagen I (Col I)-mRNA expression in mice lacking the AT2 receptor (AT2 / ) vs. wildtype (AT2 +/+) after autoradiographic signal analysis. Data are shown as mean F S.D. in PSL/ mm2 after normalization to rL32-mRNA levels (n = 6 per group); *P < 0.05.
tion assay for AT2-deficient mice (Fig. 6). As indicated by Sirius red staining on protein level, the collagen ImRNA concentrations are significantly lower in these AT2 knockouts compared to their wildtype controls ( P < 0.05).
4. Discussion This is the first description of cardiac BNP expression in mice genetically altered for components of the RAS. Taking the results from angiotensinogen-overexpression and AT1Adeficient animals, our data would clearly indicate the linearity between the level of Ang II-mediated stimulation of the AT1 receptor and cardiac BNP expression. Nevertheless, these data could not distinguish between direct peptide effects or secondary effects mediated by blood pressure alterations. Since we demonstrate that normotensive mice lacking the AT2 receptor [16,22,23] show also an impaired cardiac BNP expression, we can exclude an exclusive influence of the blood pressure and have to assume a direct effect of the cardiac RAS on BNP expression. The existence of a blood pressure-independent factor is also indicated by our observation that hypotensive angiotensinogen-deficient animals show unchanged cardiac BNP-mRNA levels. There seems to be a discrepancy between the results of both
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hypotensive animal models (angiotensinogen- or AT1-deficient mice), since in both models the Ang II/AT1-axis is disrupted which should lead to the same regulation of BNP. We have to consider that the lack of the AT1 receptor could cause an additional stimulation of the AT2 receptor and an increase in other cardiovascular active Ang metabolites, whereas the angiotensinogen-deficient animals also lack the effects of AT2 stimulation such as Ang IV and Ang(1-7) signaling. This is of particular interest, since Ang-(1-7) has been characterized as a cardioprotective factor [24]. Additionally, the difference could be also mediated by a blocked Ang II-AT1B axis in angiotensinogen-deficient animals, whereas the AT1B is still present in AT1A knockouts. If elevated BNP is a marker for cardiac failure [8,12] and the angiotensinogen-deficient animals have higher BNP-mRNA concentrations compared to AT1 knockouts, we have to conclude that the selective deletion of the ‘‘bad’’ angiotensin receptor AT1 is cardioprotective compared to the disruption of the whole RAS in angiotensinogen-deficient mice where also positive components like Ang-(1-7) are switched off. Secondly, Sakata et al. [6] conclude also in their paper that the BNP increase is directly controlled by the AT1 receptor. This would confirm our data of dramatic reduction in BNP expression in animals lacking this receptor. Therefore, further cell culture experiments have to clarify, whether the treatment of cardiomyocytes with Ang II would lead to a direct up-regulation of BNP mRNA or whether the observed reduction of BNP after AT1 blockade is mediated indirectly by an improved cardiac function. What other factors than blood pressure may trigger cardiac BNP-mRNA expression? Firstly, cardiac fibrosis is associated with elevated BNP concentrations in the heart [25]. Mice deficient for the BNP gene (NppB knockout) indicate that this elevation is not just a marker of fibrotic status. These animals are characterized by normotension but a rise in cardiac fibrosis which indicates that BNP acts locally as an antifibrotic factor in the heart [26,27] concluding that high BNP in the state of fibrosis is a cardiac rescue mechanism to prevent further deterioration. Our group recently demonstrated that increased fibrosis in angiotensinogen-overexpressing mice correlated with a rise in BNP mRNA [28]. Since our present data clearly demonstrate that reduced fibrosis correlates to a lower BNP-mRNA concentration in normotensive AT2-deficient animals and that there is no change in fibrosis or BNP mRNA in hypotensive angiotensinogen-deficient mice, it is fibrosis rather than blood pressure that regulates cardiac BNP expression. This postulate is also supported by findings of Sakata et al. [6] demonstrating that BNP-gene expression is not enhanced by initial compensatory hypertrophy but is enhanced by left ventricular fibrosis. Following this argumentation, the reduction of BNP mRNA in normotensive AT2-knockout animals implies a profibrotic function of this receptor that is blocked by AT2 deficiency. This is supported by the observation of Ichihara et al. [29] who demonstrated that animals
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with the genetic deletion of the AT2 receptor do not develop cardiac fibrosis in Ang II-induced chronic hypertension. In summary, the observed alterations in fibrosis and the unchanged BNP in hypotensive angiotensinogen knockouts and impaired BNP-mRNA expression in normotensive AT2-deficient mice demonstrate a direct influence of components of the RAS on the expression of BNP. This interaction is mediated more by fibrosis than blood pressure changes.
Acknowledgements The study was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG; WA-1441/1). We thank the Humboldt Foundation for the post-doctoral fellowship to S. H-W.
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