NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function
2003; Springer Nature; Volume: 22; Issue: 23 Linguagem: Inglês
10.1093/emboj/cdg601
ISSN1460-2075
Autores Tópico(s)RNA modifications and cancer
ResumoArticle1 December 2003free access NRSF regulates the fetal cardiac gene program and maintains normal cardiac structure and function Koichiro Kuwahara Koichiro Kuwahara Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yoshihiko Saito Corresponding Author Yoshihiko Saito Present address: 1st Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara-city Nara, 634-8522 Japan Search for more papers by this author Makoto Takano Makoto Takano Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, 606-8501 Japan Search for more papers by this author Yuji Arai Yuji Arai Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan Search for more papers by this author Shinji Yasuno Shinji Yasuno Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yasuaki Nakagawa Yasuaki Nakagawa Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Nobuki Takahashi Nobuki Takahashi Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yuichiro Adachi Yuichiro Adachi Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Genzo Takemura Genzo Takemura Department of Second Internal Medicine, Gifu University, Gifu, 500-8705 Japan Search for more papers by this author Minoru Horie Minoru Horie Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yoshihiro Miyamoto Yoshihiro Miyamoto Division of Atherosclerosis and Diabetes Mellitus, National Cardiovascular Center, Suita, Osaka, 565-8565 Japan Search for more papers by this author Takayuki Morisaki Takayuki Morisaki Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan Search for more papers by this author Shinobu Kuratomi Shinobu Kuratomi Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, 606-8501 Japan Search for more papers by this author Akinori Noma Akinori Noma Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, 606-8501 Japan Search for more papers by this author Hisayoshi Fujiwara Hisayoshi Fujiwara Department of Second Internal Medicine, Gifu University, Gifu, 500-8705 Japan Search for more papers by this author Yasunao Yoshimasa Yasunao Yoshimasa Division of Atherosclerosis and Diabetes Mellitus, National Cardiovascular Center, Suita, Osaka, 565-8565 Japan Search for more papers by this author Hideyuki Kinoshita Hideyuki Kinoshita Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Rika Kawakami Rika Kawakami Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Ichiro Kishimoto Ichiro Kishimoto Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Michio Nakanishi Michio Nakanishi Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Satoru Usami Satoru Usami Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yoshitomo Saito Yoshitomo Saito Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Masaki Harada Masaki Harada Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Kazuwa Nakao Kazuwa Nakao Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Koichiro Kuwahara Koichiro Kuwahara Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yoshihiko Saito Corresponding Author Yoshihiko Saito Present address: 1st Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara-city Nara, 634-8522 Japan Search for more papers by this author Makoto Takano Makoto Takano Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, 606-8501 Japan Search for more papers by this author Yuji Arai Yuji Arai Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan Search for more papers by this author Shinji Yasuno Shinji Yasuno Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yasuaki Nakagawa Yasuaki Nakagawa Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Nobuki Takahashi Nobuki Takahashi Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yuichiro Adachi Yuichiro Adachi Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Genzo Takemura Genzo Takemura Department of Second Internal Medicine, Gifu University, Gifu, 500-8705 Japan Search for more papers by this author Minoru Horie Minoru Horie Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yoshihiro Miyamoto Yoshihiro Miyamoto Division of Atherosclerosis and Diabetes Mellitus, National Cardiovascular Center, Suita, Osaka, 565-8565 Japan Search for more papers by this author Takayuki Morisaki Takayuki Morisaki Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan Search for more papers by this author Shinobu Kuratomi Shinobu Kuratomi Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, 606-8501 Japan Search for more papers by this author Akinori Noma Akinori Noma Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, 606-8501 Japan Search for more papers by this author Hisayoshi Fujiwara Hisayoshi Fujiwara Department of Second Internal Medicine, Gifu University, Gifu, 500-8705 Japan Search for more papers by this author Yasunao Yoshimasa Yasunao Yoshimasa Division of Atherosclerosis and Diabetes Mellitus, National Cardiovascular Center, Suita, Osaka, 565-8565 Japan Search for more papers by this author Hideyuki Kinoshita Hideyuki Kinoshita Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Rika Kawakami Rika Kawakami Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Ichiro Kishimoto Ichiro Kishimoto Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Michio Nakanishi Michio Nakanishi Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Satoru Usami Satoru Usami Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Yoshitomo Saito Yoshitomo Saito Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Masaki Harada Masaki Harada Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Kazuwa Nakao Kazuwa Nakao Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan Search for more papers by this author Author Information Koichiro Kuwahara1, Yoshihiko Saito 2, Makoto Takano3, Yuji Arai4, Shinji Yasuno1, Yasuaki Nakagawa1, Nobuki Takahashi1, Yuichiro Adachi1, Genzo Takemura5, Minoru Horie6, Yoshihiro Miyamoto7, Takayuki Morisaki4, Shinobu Kuratomi3, Akinori Noma3, Hisayoshi Fujiwara5, Yasunao Yoshimasa7, Hideyuki Kinoshita1, Rika Kawakami1, Ichiro Kishimoto1, Michio Nakanishi1, Satoru Usami1, Yoshitomo Saito1, Masaki Harada1 and Kazuwa Nakao1 1Department of Medicine and Clinical Science, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan 2Present address: 1st Department of Internal Medicine, Nara Medical University, 840 Shijo-cho, Kashihara-city Nara, 634-8522 Japan 3Department of Physiology and Biophysics, Kyoto University Graduate School of Medicine, Kyoto, 606-8501 Japan 4Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka, 565-8565 Japan 5Department of Second Internal Medicine, Gifu University, Gifu, 500-8705 Japan 6Department of Cardiovascular Medicine, Kyoto University Graduate School of Medicine, Kyoto, 606-8507 Japan 7Division of Atherosclerosis and Diabetes Mellitus, National Cardiovascular Center, Suita, Osaka, 565-8565 Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6310-6321https://doi.org/10.1093/emboj/cdg601 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Reactivation of the fetal cardiac gene program is a characteristic feature of hypertrophied and failing hearts that correlates with impaired cardiac function and poor prognosis. However, the mechanism governing the reversible expression of fetal cardiac genes remains unresolved. Here we show that neuron-restrictive silencer factor (NRSF), a transcriptional repressor, selectively regulates expression of multiple fetal cardiac genes, including those for atrial natriuretic peptide, brain natriuretic peptide and α-skeletal actin, and plays a role in molecular pathways leading to the re-expression of those genes in ventricular myocytes. Moreover, transgenic mice expressing a dominant-negative mutant of NRSF in their hearts exhibit dilated cardiomyopathy, high susceptibility to arrhythmias and sudden death. We demonstrate that genes encoding two ion channels that carry the fetal cardiac currents If and ICa,T, which are induced in these mice and are potentially responsible for both the cardiac dysfunction and the arrhythmogenesis, are regulated by NRSF. Our results indicate NRSF to be a key transcriptional regulator of the fetal cardiac gene program and suggest an important role for NRSF in maintaining normal cardiac structure and function. Introduction Cardiac hypertrophy is initially an adaptive response of the heart to mechanical stress, tissue injury or neurohumoral activation; however, sustained hypertrophy can lead to dilated cardiomyopathy and heart failure. Up to 50% of the deaths among heart failure patients are sudden and unexpected, and are presumably the result of lethal arrhythmias (Tomaselli and Marbán, 1999). The dysregulation of a panel of cardiac genes accounts for the biochemical, structural, functional and electrical alterations in failing hearts, but relatively little is known about the molecular pathways underlying those complex remodeling processes. One of the characteristic genetic alterations in hypertrophied and failing hearts is reactivation of a fetal cardiac gene program, i.e. upregulation of genes encoding atrial and brain natriuretic peptides (ANP and BNP, respectively), as well as fetal contractile protein isoforms such as β-myosin heavy chain (MHC) and α-skeletal actin (Chien et al., 1991). Indeed, production of ANP and BNP in cardiomyocytes is markedly augmented in hypertrophied and failing hearts and is a prognostic indicator of clinical severity (Kjær and Hesse, 2001). Accordingly, elucidation of the regulatory mechanisms governing expression of these fetal cardiac genes should enable one to understand better the processes by which cardiac hypertrophy and heart failure are established. Altered expression of certain ion channels in diseased hearts is also indicative of the reactivation of a fetal gene program. Two ionic currents, the hyperpolarization activated non-selective cation current (If) and the T-type Ca2+ current (ICa,T), which are normally expressed in fetal ventricles but repressed in adult ventricles, are re-expressed in ventricular myocytes of hypertrophied or failing hearts, perhaps increasing the vulnerability of the hearts to ventricular arrhythmias (Nuss and Houser, 1993; Sen and Smith, 1994; Cerbai et al., 1996, 2001; Martinez et al., 1999). Moreover, increased expression of ICa,T might contribute to the progression of heart failure (Clozel et al., 1999). Thus, elucidation of the mechanisms that control the fetal cardiac gene program may also shed light on the molecular basis for arrhythmias and sudden death associated with heart failure. Despite considerable effort, virtually the entire process by which reversible expression of fetal cardiac genes is governed remains unknown. It was shown recently that (i) a repressor element named neuron-restrictive silencer element (NRSE), also known as repressor element-1 (RE-1) (Kraner et al., 1992; Mori et al., 1992), is present in the 3′-untranslated region (UTR) of ANP; (ii) NRSE represses basal expression of ANP in ventricular myocytes by recruiting the transcriptional repressor neuron-restrictive silencer factor (NRSF), also known as RE-1 silencing transcription factor (REST) (Schoenherr and Anderson, 1995; Chong et al., 1995), which forms a complex with histone deacetylases (HDACs); and (iii) attenuation of NRSE-mediated repression is an important component of the signaling pathways by which endothelin (ET)-1 induces ANP promoter activity (Kuwahara et al., 2001). We also showed that NRSE in the 5′-flanking region of BNP represses transcription of BNP and that attenuation of NRSE-mediated repression contributes to the increase in BNP transcription induced by hypertrophic stimuli (Ogawa et al., 2002). Notably, NRSE is also reportedly present in the α-skeletal actin gene (Schoenherr et al., 1996). The aforementioned findings suggest that the NRSE–NRSF system regulates the expression of multiple fetal cardiac genes. However, the role of the NRSE–NRSF system in the expression of endogenous fetal cardiac genes and in the regulation of cardiac function remains unknown. To address these questions, in this study, we used a recombinant adenovirus expressing a dominant-negative mutant of NRSF (dnNRSF) and transgenic mice expressing dnNRSF in their hearts. We show that the inhibition of NRSF induces endogenous expression of multiple fetal cardiac genes in ventricular myocytes and markedly reduces the response of fetal cardiac genes to hypertrophic stimuli both in vitro and in vivo, suggesting that NRSF-mediated repression contributes to the dynamic regulation of expression of fetal cardiac genes, and that persistent inhibition of cardiac NRSF repressor function leads to dilated cardiomyopathy and sudden death. We also show that the genes encoding the ion channels that carry If and ICa,T are upregulated in the ventricles of the transgenic mice, and that they are downstream targets of NRSF. Taken together, our findings indicate that NRSF is a novel and important regulator of the fetal cardiac gene program responsible for maintaining normal cardiac structure and function, and that NRSF plays a key role in mediating signaling pathways that lead to heart failure and sudden cardiac death. Results NRSF regulates endogenous cardiac fetal gene expression in vitro and in vivo We previously used promoter–reporter constructs to show that the NRSF–NRSE system represses transcription of ANP and BNP in cultured ventricular myocytes. Moreover, the fact that NRSE is also present in the α-skeletal actin gene suggests that NRSF represses expression of multiple fetal cardiac genes (Schoenherr et al., 1996; Kuwahara et al., 2001; Ogawa et al., 2002). To confirm the function of NRSF in the regulation of endogenous fetal cardiac gene expression, we infected cultured ventricular myocytes with a recombinant adenovirus expressing dnNRSF (Ad/dnNRSF) and examined the resultant gene expression. dnNRSF contains a DNA-binding domain but lacks two identified repressor domains, and thus inhibits NRSE-mediated repression (Chen et al., 1998). We previously confirmed that this dnNRSF construct removes the repression of NRSE-containing reporter constructs exclusively in cultured ventricular myocytes (Kuwahara et al., 2001; Ogawa et al., 2002). Endogenous expression of ANP, BNP and α-skeletal actin mRNA, but not α-cardiac actin or GAPDH mRNA was markedly increased in ventricular myocytes infected with Ad/dnNRSF, as compared with cells infected with control vector (Figure 1A). Figure 1.NRSF regulates expression of endogenous ANP, BNP and α-skeletal actin in ventricular myocytes. (A) Northern blots showing levels of ANP, BNP, α-skeletal actin (SkA), α-cardiac actin (CaA) and GAPDH mRNA in cultured ventricular myocytes infected with Ad/lacZ or Ad/dnNRSF for 24 h. (B) A cDNA construct for the generation of dnNRSF Tg mice; Hgh, the poly(A) sequence of human growth hormone gene. (C) Western blot analysis for dnNRSF expression in ventricles from NTg and two different founders lines of dnNRSF Tg mice (Tg471 and 474). (D) Western blot analysis for dnNRSF expression in various organs from dnNRSF Tg mice: H, heart; Lu, lung; L, liver; K, kidney. (E) Representative northern blots showing levels of the indicated mRNAs in dnNRSF Tg and NTg hearts. (F) Bar graph summarizing the relative cardiac levels of the indicated mRNAs (normalized to GAPDH mRNA levels) detected in the northern blots shown in (E); bars represent means ± SEM from three independent experiments; *P < 0.05 versus NTg hearts; n = 6 each. (G) RT–PCR analysis for NRSF and ANP mRNA expression in ventricles from 13.5-day mouse embryos (ED 13.5) and 20-week-old adult mice (AD). (H) Bar graphs showing relative NRSF mRNA levels (normalized to GAPDH mRNA levels) determined by quantitative RT–PCR. Bars represent means ± SEM from three independent assays; *P < 0.05 versus ED13.5. Download figure Download PowerPoint Because mice lacking NRSF die in utero, it is impossible to use these animals to analyze NRSF function in the post-natal heart (Chen et al., 1998). However, expression of dnNRSF in chick embryo using a retroviral vector caused ectopic expression of a specific set of neuronal genes, as did targeted deletion of NRSF in mice (Chen et al., 1998). Therefore, to investigate the role of NRSF in the post-natal heart, we produced transgenic mice that express dnNRSF under the control of the cardiac-specific α-MHC promoter (Figure 1B). Two independently derived dnNRSF transgenic (Tg) founders (Tg471 and Tg474; dnNRSF mRNAs were expressed at levels 35.5- and 27.0-fold higher than endogenous NRSF mRNAs, respectively) were obtained and investigated (Figure 1C). The data obtained from Tg471, which are essentially identical to those obtained from Tg474, are presented below. Western blot analysis confirmed that expression of dnNRSF was restricted to the heart, though weak expression was observed in the lung, where endogenous α-MHC is expressed in cardiac myocytes surrounding pulmonary veins (Figure 1D) (Subramaniam et al., 1991). To determine the role of NRSF in the regulation of fetal cardiac gene expression in vivo, we carried out northern blot analyses of the genes for ANP, BNP, α-skeletal actin, β-MHC, α-cardiac actin, myosin light chain (MLC)-2v, sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) 2 and GAPDH using total RNA prepared from ventricles taken from 8- and 20-week-old mice (Figure 1E and F). The expression of the genes for ANP, BNP and α-skeletal actin, which contain an NRSE, was selectively upregulated in the hearts of dnNRSF Tg mice by 8 weeks of age. In 8-week-old dnNRSF Tg and non-transgenic (NTg) mice, the expression of another fetal gene, β-MHC, which does not contain an NRSE, was not enhanced, and the heart to body weight ratios and cardiac function estimated by echocardiography and hemodynamic analysis were not different. Apparently, induction of these fetal genes in 8-week-old dnNRSF Tg hearts is a primary effect of NRSF inhibition (Figures 1E and F, and 3B and C and Table I). Taken together, these results and the data obtained from in vitro studies using cultured ventricular myocytes, which are not affected by hemodynamic alterations, clearly indicate that NRSF regulates expression of multiple fetal cardiac genes in ventricular myocytes. Table 1. Echocardiographic and hemodynamic analysis at 8 or 20 weeks of age 8 weeks 20 weeks NTg Tg NTg Tg Echocardiographic data n = 6 n = 6 n = 5 n = 5 LVDd (mm) 4.02 ± 0.11 4.00 ± 0.29 4.04 ± 0.23 5.32 ± 0.22a LVDs (mm) 2.76 ± 0.14 2.87 ± 0.20 2.58 ± 0.15 4.66 ± 0.15a IVST (mm) 0.66 ± 0.038 0.65 ± 0.028 0.58 ± 0.08 0.60 ± 0.06 PWT (MM) 0.67 ± 0.042 0.69 ± 0.034 0.60 ± 0.07 0.54 ± 0.05 FS (%) 32.8 ± 1.88 27.9 ± 1.54 36.2 ± 2.08 12.0 ± 0.84a EF (%) 69.7 ± 2.56 62.67 ± 2.20 71.8 ± 2.75 30.4 ± 1.94 Hemodynamic data n = 4 n = 5 n = 6 n = 5 dP/dtmax (mmHg/s) 4865 ± 201 4757 ± 325 4090 ± 188 2328 ± 264a dP/dtmin (mmHg/s) −4935 ± 218 −4756 ± 237 −4150 ± 193 −2264 ± 292a HR (/min) 498 ± 26.9 533 ± 29.4 503 ± 42.8 487 ± 43.6 LVSP (mmHg) 98.9 ± 4.53 95.1 ± 3.6 88.4 ± 3.54 68.3 ± 5.34a LVEDP (mmHg) 2.25 ± 0.56 2.36 ± 0.69 3.83 ± 1.04 7.68 ± 2.30 Values are means ± SEM. LVDd, left ventricular end diastolic dimension; LVDs, left ventricular end systolic dimension; IVST, interventricular septal thickness; PWT, posterior wall thickness; FS, fractional shortening; EF, ejection fraction; dP/dt, first derivative of pressure; HR, heart rate; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end diastolic pressure. a P < 0.05 dnNRSF Tg versus NTg mice. Several earlier studies reported that expression of NRSF mRNA is barely detectable in fetal hearts, though its expression is detected in neonatal ventricular myocytes and adult hearts (Schoenherr and Anderson, 1995; Chong et al., 1995; Palm et al., 1998; Grimes et al., 2000; Kuwahara et al., 2001). We therefore used RT–PCR to examine expression of NRSF and ANP in fetal ventricles obtained from 13.5-day mouse embryos and adult ventricles from 20-week-old mice. Consistent with those earlier reports, expression of NRSF mRNA was significantly lower in the embryonic than adult ventricles. Conversely, expression of ANP mRNA was significantly lower in adult ventricles than embryonic ventricles (Figure 1G). These results, which were confirmed by quantitative real-time RT–PCR (Figure 1H), suggest that NRSF plays a role in the developmental regulation of fetal cardiac gene expression and further support the notion that NRSF represses expression of fetal cardiac genes. NRSF plays an important role in the re-expression of fetal cardiac genes induced by hypertrophic stimuli To examine further the role of NRSF in the re-expression of endogenous fetal cardiac genes in vivo, we subjected the hearts of dnNRSF Tg mice and their NTg littermates to acute pressure overload created by transverse aortic constriction (TAC). As previously reported (Harada et al., 1998), acute pressure overload induced expression of c-fos and BNP in NTg hearts within 30 min after the onset of TAC (Figure 2A–C). Northern blot analysis showed that basal levels of BNP expression were higher in 8-week-old dnNRSF Tg than NTg hearts, but that induction of BNP expression by TAC was less pronounced in dnNRSF Tg hearts (Figure 2A and B). In contrast, the inducibility of c-fos expression and peak-to-peak systolic pressure gradients across the stenosis were similar in dnNRSF Tg and NTg hearts (Figure 2A, C and D). These results were confirmed by quantitative RT–PCR analysis for BNP mRNA expression (Figure 2E) and indicate that BNP expression is constitutively elevated in hearts expressing dnNRSF, though its inducibility is diminished. Figure 2.NRSF regulates the inducible response of cardiac fetal gene expression to hypertrophic stimuli. (A) Northern blots showing levels of BNP, c-fos and GAPDH mRNA in sham- or TAC-operated NTg and dnNRSF Tg hearts. (B and C) Bar graphs summarizing the fold increases in BNP (B) and c-fos (C) mRNA detected in the northern blots shown in (A); n = 4 each. (D and E) Pressure gradients across the stenosis after TAC (D) and quantitative real-time RT–PCR analyses for BNP mRNA expression (E); n = 4 each. (F) Representative northern blots showing levels of ANP, BNP, SkA and GAPDH mRNA in cultured ventricular myocytes infected with Ad/lacZ or Ad/dnNRSF and subsequently treated for 24 h with or without 10 nM ET-1. (G) Bar graphs summarizing the percentage increases in the levels of ANP, BNP and SkA mRNA elicited by 10 nM ET-1 detected in the northern blots shown in (F); *P < 0.05 versus Ad/lacZ; n = 5 each. (H) Bar graph summarizing the results of a quantitative real-time RT–PCR analysis of ANP mRNA expression; *P < 0.01, P < 0.05; n = 4 each. The numbers above each bar indicate the fold increase induced by ET-1. For (B), (C), (E), (G) and (H), relative mRNA levels (normalized to GAPDH mRNA levels) are presented as means ± SEM. (I) Surface areas of 40 randomly selected cells in each group normalized to control myocytes (=100%) (mean ± SEM); *P < 0.05; NS, not significant. (J) The promoter activities of 5UAS-hANPLuc in ventricular myocytes treated or not with 10 nM ET-1 for 48 h in the absence or presence of a GAL4-NRSF fusion protein. The activities of the construct transfected into myocytes treated with 10 nM ET-1 are expressed as the fold increase over the value for myocytes treated without ET-1 in the right panel. Bars represent means ± SEM of the relative luciferase activity from at least three separate assays carried out in triplicate; *P < 0.05 versus GAL4-NRSF(−) ET-1(−); †P < 0.05. (K) Heart weight to body weight ratios in sham- and TAC-operated mice. Bars represent means ± SEM. (L and M) Quantitative RT–PCR analysis of BNP (L) and NRSF (M) mRNA levels. Relative mRNA levels (normalized to GAPDH mRNA levels) are presented as means ± SEM; levels in sham-operated mice are assigned a value of 1.0; *P < 0.05 versus sham-operated mice. (N) Inverse correlation between expression of ventricular BNP and NRSF mRNAs. Download figure Download PowerPoint Figure 3.Dilated cardiomyopathy in dnNRSF Tg mice. (A) Kaplan–Meier survival analysis of dnNRSF Tg471 (n = 68), Tg474 (n = 35) and NTg (n = 145) mice showing a significant difference in survival rates between dnNRSF Tg and NTg mice (log rank test; P < 0.0001). (B) Hearts of NTg and dnNRSF Tg mice at 8, 12 and 20 weeks; scale bars, 10 mm. (C) Heart to body weight ratios (mg/g), (D) lung to body weight ratios (mg/g) and (E) body weights (g) in NTg and dnNRSF Tg mice measured at the indicated times; *P < 0.05; NS, not significant. (F) Representative M-mode echocardiographic tracings from 20-week-old NTg and dnNRSF Tg mice: IVS, interventricular septum; LV; left ventricle; PW, posterior wall; ESD, end systolic dimension; EDD, end diastolic dimension. (G) Representative high fidelity left ventricular pressure tracings from 20-week-old NTg and dnNRSF Tg mice: HR, heart rate; LVP, LV pressure. (H–Q) Histological analysis of hearts from 20-week-old NTg and dnNRSF Tg mice. (H and I) Hearts from NTg (H) and dnNRSF Tg (I) mice were sectioned longitudinally and stained with HE; scale bars, 2.5 mm. (J–M) Photomicrographs of histological sections of left ventricle from NTg (J and L) and dnNRSF Tg mice (K and M) stained with Masson's trichrome stain; collagen stains blue with trichrome stain (M); scale bars, 20 μm. (N–Q) Transmission electron micrographs of cardiac myocytes from 20-week-old NTg (N and O) and dnNRSF Tg (P and Q) mice: arrows indicate disrupted mitochondria; My, myofibrils; Mt, mitochondria; N, nucleus; scale bars, 1 μm. Download figure Download PowerPoint Consistent with that finding, expression of dnNRSF markedly reduced the fold increases in endogenous ANP, BNP and α-skeletal actin expression induced by ET-1 in cultured ventricular myocytes (Figure 2F–H), whereas it had no significant effect on ET-1-induced increases in cell size (Figure 2I). In addition, when we incubated ventricular myocytes co-transfected with an ANP promoter–reporter construct containing GAL4-binding sites (5UAS-hANPLuc) and a plasmid encoding a GAL4 DNA-binding domain–NRSF fusion protein (GAL4-NRSF) with ET-1, we found that in the presence of GAL4-NRSF, the response of 5UAS-hANPLuc to ET-1 was significantly increased, though the basal control activity of 5UAS-hANPLuc was significantly reduced (Figure 2J). This means that NRSF-mediated repression is necessary for full induction of fetal cardiac gene expression du
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