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Critical Role of the Transcriptional Repressor NRSF System in AngiotensinII-induced Aldosterone Synthesis in Human Adrenocortical Cells(H295R)
S164 Journal of Cardiac Failure Vol. 12 No. 8 Suppl. 2006 gene transfer to compensate reduced expression of the gene in the rat ischemic heart failure model. Results: The therapeutic effect of lenti-SERCA2 vector (1x1011 IU/300g BW) was compared with the lenti-b-Gal control gene injection on 6 months after gene transfer. Echocardiography revealed that lenti-SERCA2 gene transfer significantly prevented an increase in LV diameter and a decrease of fractional shortening (lenti-SERCA2: 16.262.5% vs. lenti-b-Gal: 10.262.4%). Pressure-volume loop analysis demonstrated that lenti-SERCA2 introduction improved both systolic and diastolic function. SERCA2 protein levels were elevated and the BNP mRNA expression was significantly decreased in lenti-SERCA2 group. Furthermore, DNA microarray disclosed that SERCA2 gene transfer increased genes for sarcomeric proteins, mitochondria, SOD, catalase, a-adrenergic receptor, adenylyl cyclase, PI3-kinase, Akt, calcineurin, but decreased genes for fibrosis, caspase 3, TNF-a and its receptor, endothelin-1, angiotensin II type I receptor. Conclusion: Our study showed that the SERCA2 gene was successfully integrated into the host heart, induced favorable molecular remodeling, prevented a mal-remodeling and then improved the survival rate in the ischemic cardiomyopathy model. These results support the premise that a strategy to consistently compensate the reduced SERCA2 gene expression improves human heart failure.
1016 Bi-directional N-terminal and Central Domain Interactions as a Critical Mechanism for Channel Gating of Ryanodine Receptor HIROKI TATEISHI, MASAFUMI YANO, TAKESHI YAMAMOTO, XIAOJUAN XU, HITOSHI UCHINOUMI, MAMORU MOCHIZUKI, TOSHIYUKI NOMA, SHIGEKI KOBAYASHI, TOMOKO OHKUSA, MASUNORI MATSUZAKI Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, Ube, Japan DPc10, a synthetic peptide (Gly2460-Pro2495) of RyR2 (one of the mutable domains in CPVT), mimics channel dysfunction in human CPVT, by acting competitively to reduce stabilizing interactions between the N-terminal1-600 and central2000-2500 mutable domains of RyR2. Here, using this domain peptide approach, we assessed the role of the N-terminal and central mutable domain interactions on Ca2 þ release function of RyR2.SR vesicles were isolated from dog LV muscles (n55), then RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) using N-terminal or central domain peptide (DP163-195 or DP2460-2495), which includes single point mutation site in ARVC or CPVT, respectively, as a carrier. Both DP163-195 and DP2460-2495 mediated a specific MCA fluorescence labeling of RyR2. Incorporation of either DP163-195 or DP2460-2495 to normal cardiomyocytes (by protein delivery kit) induced diastolic Ca2 þ spark, and delayed afterdepolarization-mediated Ca2 þ transient. However, either Arg-to-Gln mutation (DP163-195mut) or Arg-to-Ser mutation (DP2460-2495mut) made in these domain peptides, mimicking the same human mutation in ARVC or CPVT, abolished all of these effects that would have been produced by these domain peptides. Thus, there is a tight interaction between N-terminal and central domains, in which several human ARVC or CPVT mutation sites are included. Either specific mutation of N-terminal or central domain leads to common defective inter-domain interaction, resulting in diastolic Ca2 þ spark that may trigger ventricular arrhythmia.
Ca2þ spark frequency (s-1100microm-1) was markedly increased (4.261.6, p!0.01) compared with normal cardiomyocytes (1.460.5). Incorporation of DPc2114-2149 (by protein delivery kit) markedly decreased the frequency of Ca2þ spark (1.660.7). A single Val-to-Met mutation made in DPc2114-2149, mimicking MH mutation, abolished all of these effects that would have been produced by DP2114-2149. Thus, specific inter-domain interaction including domain2114-2149 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure.
1018 Critical Role of the Transcriptional Repressor NRSF System in AngiotensinIIinduced Aldosterone Synthesis in Human Adrenocortical Cells(H295R) SATOSHI SOMEKAWA, IMAGAWA KEIICHI, NAYA NORIYUKI, UEMURA SHIRO, SAITO YOSHIHIKO The 1st Department of Internal Medicine, Nara Medical University, Kashihara, Japan Background and Aim: Aldosterone is synthesized by CYP11B2 synthase, of which expression is regulated by Ca2þ influx through calcium channel in response to angiotensinII (AngII). Recently we found that inhibition of neuron restrictive silencer factor (NRSF), which binds to neuron restrictive silencer element (NRSE) and suppresses NRSE-containing genes, is involved in re-induction of cardiac embryonic genes. Computer analysis revealed that NRSE is contained CYP11B2 gene and CACNA1H gene that encodes subunit of Cav3.2. The aim of study is to elucidate the roles of NRSE/NRSF system on aldosterone synthesis. Methods and Result: Inhibition of endogenous NRSF function by dominant-negative NRSF adenovirus (AD/dnNRSF) markedly increased aldosterone secretion and CYP11B2 mRNA. AD/dnNRSF also increased CACNA1H mRNA. In the absence of AD/dnNRSF, AngII increased CYP11B2 mRNA by 18.3-fold, but in the presense of AD/dnNRSF AngII increased it by only 1.5-fold. Next, we subcloned NRSE or mutated NRSE (mtNRSE) into downstream of a luciferase gene driven by CYP11B2 promoter. Co-transfection of dnNRSF with these reporter genes showed 4.4-fold increase in the luciferase activity of CYP11B2/luc/NRSE and 2.4-fold increase of the luciferase activity of CYP11B2/luc/mtNRSE, suggesting the involvement of indirect effect of another NRSE-containing gene, CACNA1H. Conclusion: These findings suggest that NRSE/NRSF system is involved in basal and AngII-induced expression of CYP11B2. This system plays a key role in the aldosterone synthesis through gene expression of CYP11B2 and CACNA1H.
1019 PPAR-gamma Agonist Suppresses 11beta-hydroxysteroid Dehydrogenase Type 1 Expression and Alleviates Glucocorticoid-induced ACE Gene Expression in Cardiac Fibroblasts UEHARA YOSHIKI1, AZUMA YOSHIYUKI2, MINAI KOUSUKE2, SHIMIZU MITSUYUKI1, MOCHIZUKI SEIBU3 1 Division of Cardiology, Department of Internal Medicine, The Jike University School of Medicine Kashiwa Hospital, Kashiwa, Japan, 2Institute of Clinical Medicine and Research, The Jike University School of Medicine Kashiwa Hospital, Kashiwa, Japan, 3Division of Cardiology, Department of Internal Medicine, The Jike University School of Medicine, Tokyo, Japan
1017 Specific Inter-domain Interaction within Ryanodine Receptor as a new Therapeutic Target in Heart Failure TAKESHI YAMAMOTO, MASAFUMI YANO, XU XIAJUAN, HITOSHI UCHINOUMI, HIROKI TATEISHI, MAMORU MOCHIZUKI, SHIGEKI KOBAYASHI, MASUNORI MATSUZAKI Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine Interaction between N-terminal and central domains of ryanodine receptor (RyR1/ RyR2) was recently found to play a critical role in channel regulation. Here, we report that DP2114-2149, a synthetic cardiac domain peptide corresponding to Val-Met (mutable domain in malignant hyperthermia) of RyR1, shows a significant RyR2 stabilizing effect in failing hearts. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (normal: n56, 4-weeks pacing-induced heart failure: n56). RyR2 in SR was specifically MCA-labeled. After tryptic digestion, the major MCA-labeled fragment of RyR2 (145kD) was detected by an antibody (Ab) against central region (Ab2132). Addition of DP2114-2149 to MCA-labeled SR competitively induced domain unzipping between native domain2114-2149 and its counter domain (EC5050.3microM), as confirmed by quenching of the MCA fluorescence by a large-size fluorescence quencher. The DP2114-2149 prevented spontaneous Ca2þ leak from failing SR (IC5050.2microM). In failing cardiomyocytes, diastolic
Purpose: We previously reported corticosteroids augment the gene expression of angiotensin converting enzyme (ACE) through glucocorticoid receptor (GR) in cultured cardiac fibroblasts (CF). However, GR-active corticosterone is unable to enter the cells because of its binding to carrier protein in the serum, whereas GR-inactive 11-dehydroxycorticosterone (11-DHB) enter the cells and is converted to corticosterone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) to stimulate GR. Here, we tested the hypothesis that 11beta-HSD1 is implicated in the activation of GR in CF and determined the effect of PPAR-gamma agonist on the above action. Methods & Results: CF was prepared from neonatal rats, and mRNA expressions were analyzed using real-time RT-PCR. The basal gene expression of 11betaHSD1 was observed and 11-DHB augmented the ACE gene expression through GR. Inhibition of 11beta-HSD1 with carbenoxolone and siRNA abrogated the ACE gene expression augmented by 11-DHB, respectively. Moreover, PPAR-gamma agonist, 15-deoxy-delta12, 14-prostaglandin-J2 suppressed the 11beta-HSD1 gene expression and alleviated the 11-DHB-augmented ACE gene expression. These data indicate that 11beta-HSD1 reactivates 11-DHB at the prereceptor level to stimulate GR and augment the ACE gene expression, and that PPAR-gamma agonists regulate the 11beta-HSD1 gene expression and thus the GR-mediated ACE gene expression in CF. Conclusions: 11beta-HSD1 may play a pivotal role in the pathogenesis of heart failure and PPAR-gamma agonists are promising therapeutic reagents for heart failure.
1016 Bi-directional N-terminal and Central Domain Interactions as a Critical Mechanism for Channel Gating of Ryanodine Receptor HIROKI TATEISHI, MASAFUMI YANO, TAKESHI YAMAMOTO, XIAOJUAN XU, HITOSHI UCHINOUMI, MAMORU MOCHIZUKI, TOSHIYUKI NOMA, SHIGEKI KOBAYASHI, TOMOKO OHKUSA, MASUNORI MATSUZAKI Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine, Ube, Japan DPc10, a synthetic peptide (Gly2460-Pro2495) of RyR2 (one of the mutable domains in CPVT), mimics channel dysfunction in human CPVT, by acting competitively to reduce stabilizing interactions between the N-terminal1-600 and central2000-2500 mutable domains of RyR2. Here, using this domain peptide approach, we assessed the role of the N-terminal and central mutable domain interactions on Ca2 þ release function of RyR2.SR vesicles were isolated from dog LV muscles (n55), then RyR2 was fluorescently labeled with methylcoumarin acetate (MCA) using N-terminal or central domain peptide (DP163-195 or DP2460-2495), which includes single point mutation site in ARVC or CPVT, respectively, as a carrier. Both DP163-195 and DP2460-2495 mediated a specific MCA fluorescence labeling of RyR2. Incorporation of either DP163-195 or DP2460-2495 to normal cardiomyocytes (by protein delivery kit) induced diastolic Ca2 þ spark, and delayed afterdepolarization-mediated Ca2 þ transient. However, either Arg-to-Gln mutation (DP163-195mut) or Arg-to-Ser mutation (DP2460-2495mut) made in these domain peptides, mimicking the same human mutation in ARVC or CPVT, abolished all of these effects that would have been produced by these domain peptides. Thus, there is a tight interaction between N-terminal and central domains, in which several human ARVC or CPVT mutation sites are included. Either specific mutation of N-terminal or central domain leads to common defective inter-domain interaction, resulting in diastolic Ca2 þ spark that may trigger ventricular arrhythmia.
Ca2þ spark frequency (s-1100microm-1) was markedly increased (4.261.6, p!0.01) compared with normal cardiomyocytes (1.460.5). Incorporation of DPc2114-2149 (by protein delivery kit) markedly decreased the frequency of Ca2þ spark (1.660.7). A single Val-to-Met mutation made in DPc2114-2149, mimicking MH mutation, abolished all of these effects that would have been produced by DP2114-2149. Thus, specific inter-domain interaction including domain2114-2149 seems to mediate stabilization of RyR2 in failing hearts, which may lead to a novel therapeutic strategy against heart failure.
1018 Critical Role of the Transcriptional Repressor NRSF System in AngiotensinIIinduced Aldosterone Synthesis in Human Adrenocortical Cells(H295R) SATOSHI SOMEKAWA, IMAGAWA KEIICHI, NAYA NORIYUKI, UEMURA SHIRO, SAITO YOSHIHIKO The 1st Department of Internal Medicine, Nara Medical University, Kashihara, Japan Background and Aim: Aldosterone is synthesized by CYP11B2 synthase, of which expression is regulated by Ca2þ influx through calcium channel in response to angiotensinII (AngII). Recently we found that inhibition of neuron restrictive silencer factor (NRSF), which binds to neuron restrictive silencer element (NRSE) and suppresses NRSE-containing genes, is involved in re-induction of cardiac embryonic genes. Computer analysis revealed that NRSE is contained CYP11B2 gene and CACNA1H gene that encodes subunit of Cav3.2. The aim of study is to elucidate the roles of NRSE/NRSF system on aldosterone synthesis. Methods and Result: Inhibition of endogenous NRSF function by dominant-negative NRSF adenovirus (AD/dnNRSF) markedly increased aldosterone secretion and CYP11B2 mRNA. AD/dnNRSF also increased CACNA1H mRNA. In the absence of AD/dnNRSF, AngII increased CYP11B2 mRNA by 18.3-fold, but in the presense of AD/dnNRSF AngII increased it by only 1.5-fold. Next, we subcloned NRSE or mutated NRSE (mtNRSE) into downstream of a luciferase gene driven by CYP11B2 promoter. Co-transfection of dnNRSF with these reporter genes showed 4.4-fold increase in the luciferase activity of CYP11B2/luc/NRSE and 2.4-fold increase of the luciferase activity of CYP11B2/luc/mtNRSE, suggesting the involvement of indirect effect of another NRSE-containing gene, CACNA1H. Conclusion: These findings suggest that NRSE/NRSF system is involved in basal and AngII-induced expression of CYP11B2. This system plays a key role in the aldosterone synthesis through gene expression of CYP11B2 and CACNA1H.
1019 PPAR-gamma Agonist Suppresses 11beta-hydroxysteroid Dehydrogenase Type 1 Expression and Alleviates Glucocorticoid-induced ACE Gene Expression in Cardiac Fibroblasts UEHARA YOSHIKI1, AZUMA YOSHIYUKI2, MINAI KOUSUKE2, SHIMIZU MITSUYUKI1, MOCHIZUKI SEIBU3 1 Division of Cardiology, Department of Internal Medicine, The Jike University School of Medicine Kashiwa Hospital, Kashiwa, Japan, 2Institute of Clinical Medicine and Research, The Jike University School of Medicine Kashiwa Hospital, Kashiwa, Japan, 3Division of Cardiology, Department of Internal Medicine, The Jike University School of Medicine, Tokyo, Japan
1017 Specific Inter-domain Interaction within Ryanodine Receptor as a new Therapeutic Target in Heart Failure TAKESHI YAMAMOTO, MASAFUMI YANO, XU XIAJUAN, HITOSHI UCHINOUMI, HIROKI TATEISHI, MAMORU MOCHIZUKI, SHIGEKI KOBAYASHI, MASUNORI MATSUZAKI Division of Cardiology, Department of Medicine and Clinical Science, Yamaguchi University Graduate School of Medicine Interaction between N-terminal and central domains of ryanodine receptor (RyR1/ RyR2) was recently found to play a critical role in channel regulation. Here, we report that DP2114-2149, a synthetic cardiac domain peptide corresponding to Val-Met (mutable domain in malignant hyperthermia) of RyR1, shows a significant RyR2 stabilizing effect in failing hearts. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles (normal: n56, 4-weeks pacing-induced heart failure: n56). RyR2 in SR was specifically MCA-labeled. After tryptic digestion, the major MCA-labeled fragment of RyR2 (145kD) was detected by an antibody (Ab) against central region (Ab2132). Addition of DP2114-2149 to MCA-labeled SR competitively induced domain unzipping between native domain2114-2149 and its counter domain (EC5050.3microM), as confirmed by quenching of the MCA fluorescence by a large-size fluorescence quencher. The DP2114-2149 prevented spontaneous Ca2þ leak from failing SR (IC5050.2microM). In failing cardiomyocytes, diastolic
Purpose: We previously reported corticosteroids augment the gene expression of angiotensin converting enzyme (ACE) through glucocorticoid receptor (GR) in cultured cardiac fibroblasts (CF). However, GR-active corticosterone is unable to enter the cells because of its binding to carrier protein in the serum, whereas GR-inactive 11-dehydroxycorticosterone (11-DHB) enter the cells and is converted to corticosterone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) to stimulate GR. Here, we tested the hypothesis that 11beta-HSD1 is implicated in the activation of GR in CF and determined the effect of PPAR-gamma agonist on the above action. Methods & Results: CF was prepared from neonatal rats, and mRNA expressions were analyzed using real-time RT-PCR. The basal gene expression of 11betaHSD1 was observed and 11-DHB augmented the ACE gene expression through GR. Inhibition of 11beta-HSD1 with carbenoxolone and siRNA abrogated the ACE gene expression augmented by 11-DHB, respectively. Moreover, PPAR-gamma agonist, 15-deoxy-delta12, 14-prostaglandin-J2 suppressed the 11beta-HSD1 gene expression and alleviated the 11-DHB-augmented ACE gene expression. These data indicate that 11beta-HSD1 reactivates 11-DHB at the prereceptor level to stimulate GR and augment the ACE gene expression, and that PPAR-gamma agonists regulate the 11beta-HSD1 gene expression and thus the GR-mediated ACE gene expression in CF. Conclusions: 11beta-HSD1 may play a pivotal role in the pathogenesis of heart failure and PPAR-gamma agonists are promising therapeutic reagents for heart failure.