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Pathomorphology of Myocardium in Heart Failure
S10 Journal of Cardiac Failure Vol. 13 No. 6 Suppl. 2007
Symposium 5 S5-1 Development of Tissue- and Time-specific Gene Knockout in Mice SATOSHI SAKAI Cardiovascular Division, Graduate School of Comprehensive Human Science, University of Tsukuba, Tsukuba, Japan Gene targeting to generate loss-of-function mutations in mice has yielded remarkable advances in understanding the roles of the specific gene in development and diseases. Conventional knockout strategy has some difficulties to estimate the gene function; it leads embryonic lethality when the gene is crucial for embryogenesis and makes phenotypes hard to be analyzed since the influence of changes from embryonic stage persists. The site-specific Cre recombinase has been used to avoid these problems. It has succeeded to control the gene deletion in a tissue-specific manner by driving the Cre recombinase gene using a tissue-specific promoter; for example, alpha-myosin heavy chain promoter corresponds to cardiac myocytes. Moreover, to increase the accuracy of time-control, the Cre recombinase gene has been modified; the Cre-LBD (ligandbinding domain) system is one of ways. LBDs are derived from either the progesterone or estrogen receptor; it enables to activate the Cre recombinase by using progesterone or estrogen analogue. Thus, the conditional targeting strategy will be used further, because it makes possible to analyze in vivo functions of the target gene both tissue- and time-specifically.
S5-4 SNP Analysis TOSHIHIRO TANAKA SNP Research Center, RIKEN, Yokohama, Japan SNP is the simplest and the most common form of DNA polymorphism in the human genome. Recently, it has been widely accepted that common variant in the genome play roles in the pathogenesis of common diseases (common disease-common variant hypothesis) with increasing number of reports on genetic backgrounds of common diseases or drug sensitivity. SNPs will be a key player in personalized medicine since precise understanding of human genetic diversity is indispensable to make medicine personalized. In performing genetic association studies, first of all, one should be careful in the study design. Sensitivity and specificity of the study should be appropriately set before experiment; sample size might be one of the biggest concerns. Second, one should select genotyping method according to the sample size and the number of SNPs to be examined. Third, statistical analyses and interpretation of the results should be done carefully to identify candidate locus showing susceptibility to the disease. Fourth, by further genotyping and analyses, one should narrow down the candidate locus to single SNP or SNPs working in synergy. Lastly and hopefully, function of the SNP should be revealed to better understand the pathogenesis of the disease, thereby making anchoring point to personalized medicine.
S5-2 Pathomorphology of Myocardium in Heart Failure FUMIO TERASAKI1, YASUSHI KITAURA1, HIROAKI SHIMOMURA1, BIN TSUKADA1, SHUICHI FUJITA1, TAKASHI KATASHIMA1, KAORU OTSUKA1, KOICHI SOHMIYA1, AKIRA UKIMURA1, MASAKI IKEMOTO2, MASATOSHI FUJITA2, YUKO ITO3, YOSHINORI OTSUKI3, KYOKO IMANAKAYOSHIDA4, MICHIAKI HIROE5 1 Department of Internal Medicine III, Osaka Medical College, Takatsuki, Japan, 2 Department of Laboratory Science, School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto, Japan, 3Department of Anatomy I, Osaka Medical College, Takatsuki, Japan, 4Department of Pathology, Mie University Graduate School of Medicine, Tsu, Japan, 5Department of Nephrology and Cardiology, International Medical Center of Japan, Tokyo, Japan Histopathological examinations elucidate qualitative and quantitative structural alteration, and detect localization or expression of target substances in tissues or cells. Necrosis, apoptosis, autophagy, and abnormality in the ubiquitin-proteasome system have been proposed as the mechanisms of myocardial cell death or cell degeneration in heart failure. Moreover, inflammatory processes and extracellular matrix remodeling are important factors in the pathophysiology of heart failure. In this presentation, histological, immunohistochemical and electron-microscopic techniques for detecting and estimating these pathological conditions will be reviewed and discussed.
S5-5 Peptidome Analysis of Cardiovascular Tissue and Cells NAOTO MINAMINO, KAZUKI SASAKI Department of Pharmacology, National Cardiovascular Center Research Institute, Suita, Japan Peptides play crucial roles in many physiological events as hormones and local mediators, but the real status of peptides has not been well elucidated. This is mainly due to the facts that most peptides in the cells are degradation products of proteins, and that peptides are present in extremely low concentrations and easily susceptible to proteolysis. Recent progress in the separation and structural analysis by mass spectrometers made it possible to identify a whole set of peptides, PEPTIDOME, in the target. We started the Peptidome project in 1999 and registered peptides of the brain tissue to the Peptidome database. The quality of Peptidome data depends on that of the peptide preparation used for the analysis. However, the peptide quality obtained from cardiovascular tissue is not high in contrast with that from the endocrine tissue. We have been improving the procedures for harvesting peptides from the cardiovascular cells, and very recently succeeded in collecting relatively high quality data including known bioactive peptides. Although more trials are required to optimize the peptide quality, detailed catalogs of the peptides secreted from the cardiovascular cells will be available near in future, which allows us to develop new strategy against the cardiovascular disease.
S5-3 Noncoding RNAs, Emerging Roles in the Regulation of Cardiac Gene Expression MASASHI ARAI, ATAI WATANABE, MASAHIKO KURABAYASHI Gunma University Graduate School of Medicine, Maebashi, Japan Recently, the role of noncoding RNAs, especially small interference RNA (siRNA) and microRNA (miRNA) has been recognized. Double strand RNAs that are transcribed from genome or exogenously administered were processed by slicer enzymes, Drosha and Dicer to form 21w23 nt small RNAs. In the RNA-induced silencing complex, RISC, siRNA cut trget mRNA and miRNA suppresses the translation from target mRNA. SiRNA is especillay useful to suppress specific mRNA expression. We have demonstrated that phospholamban siRNA decreased its mRNA and protein levels to 10% of control 12 hours and 48 hours after transduction, respectively in the rat cardiac myocytes. As a result, Ca2þ uptake rate was increased by 23% estimated by its EC50 value. On the other hand, miRNA is expressed in a spacio-temporal specific manner and suppresses translation from several hundred mRNAs. miR1 decreases Hand2 protein to switch ventricular myocytes from proliferation to differentiation in the developing embryo. miR-195 is increased in the rat pressure overload cardiac hypertrophy and in human DCM heart. Overexpression of miR195 is sufficient to induce cardiac hypertrophy. Accordingly, noncoding RNAs are critically involved in the cardiac development and pathological conditions, and may serve as therapeutic targets in heart disease.
S5-6 Use of DNA Microarray for Cardiovascular Basic Research MASANORI ASAKURA1, HIROSHI ASANUMA2, JIYOONG KIM3, KAZUO KOMAMURA4, HITONOBU TOMOIKE5, MASAFUMI KITAKAZE1 1 Department of R and D of Clinical Research, National Cardiovascular Center, Suita, Japan, 2Kinki University, Osaka-Sayama, Japan, 3National Cardiovascular Center, Cardiovascular Division, Suita, Japan, 4National Cardiovascular Center Research Institute, Suita, Japan, 5National Cardiovascular Center, Suita, Japan Heart failure is one of most critical issues in cardiovascular field all over the world. Cardiologists can use several commercial drugs such as atrial natriuretic peptide, angiotensin II receptor antagonist, beta receptor blocker for the treatment of heart failure. However, most cardiologists think that current therapy for heart failure is not sufficient. It is hoped that new therapy for heart failure could be developed and be applied for the patients with heart failure. To attain this aim, it is important to explore novel targets for the treatment of heart failure. There are several approaches to search for the substances related to the pathogenesis of heart failure. These approaches include transcriptome and proteome. We have analyzed gene expression profiling of heart diseases using DNA microarray. We will present how to use DNA micorarray for the basic research on cardiovascular field, especially on heart failure.
Symposium 5 S5-1 Development of Tissue- and Time-specific Gene Knockout in Mice SATOSHI SAKAI Cardiovascular Division, Graduate School of Comprehensive Human Science, University of Tsukuba, Tsukuba, Japan Gene targeting to generate loss-of-function mutations in mice has yielded remarkable advances in understanding the roles of the specific gene in development and diseases. Conventional knockout strategy has some difficulties to estimate the gene function; it leads embryonic lethality when the gene is crucial for embryogenesis and makes phenotypes hard to be analyzed since the influence of changes from embryonic stage persists. The site-specific Cre recombinase has been used to avoid these problems. It has succeeded to control the gene deletion in a tissue-specific manner by driving the Cre recombinase gene using a tissue-specific promoter; for example, alpha-myosin heavy chain promoter corresponds to cardiac myocytes. Moreover, to increase the accuracy of time-control, the Cre recombinase gene has been modified; the Cre-LBD (ligandbinding domain) system is one of ways. LBDs are derived from either the progesterone or estrogen receptor; it enables to activate the Cre recombinase by using progesterone or estrogen analogue. Thus, the conditional targeting strategy will be used further, because it makes possible to analyze in vivo functions of the target gene both tissue- and time-specifically.
S5-4 SNP Analysis TOSHIHIRO TANAKA SNP Research Center, RIKEN, Yokohama, Japan SNP is the simplest and the most common form of DNA polymorphism in the human genome. Recently, it has been widely accepted that common variant in the genome play roles in the pathogenesis of common diseases (common disease-common variant hypothesis) with increasing number of reports on genetic backgrounds of common diseases or drug sensitivity. SNPs will be a key player in personalized medicine since precise understanding of human genetic diversity is indispensable to make medicine personalized. In performing genetic association studies, first of all, one should be careful in the study design. Sensitivity and specificity of the study should be appropriately set before experiment; sample size might be one of the biggest concerns. Second, one should select genotyping method according to the sample size and the number of SNPs to be examined. Third, statistical analyses and interpretation of the results should be done carefully to identify candidate locus showing susceptibility to the disease. Fourth, by further genotyping and analyses, one should narrow down the candidate locus to single SNP or SNPs working in synergy. Lastly and hopefully, function of the SNP should be revealed to better understand the pathogenesis of the disease, thereby making anchoring point to personalized medicine.
S5-2 Pathomorphology of Myocardium in Heart Failure FUMIO TERASAKI1, YASUSHI KITAURA1, HIROAKI SHIMOMURA1, BIN TSUKADA1, SHUICHI FUJITA1, TAKASHI KATASHIMA1, KAORU OTSUKA1, KOICHI SOHMIYA1, AKIRA UKIMURA1, MASAKI IKEMOTO2, MASATOSHI FUJITA2, YUKO ITO3, YOSHINORI OTSUKI3, KYOKO IMANAKAYOSHIDA4, MICHIAKI HIROE5 1 Department of Internal Medicine III, Osaka Medical College, Takatsuki, Japan, 2 Department of Laboratory Science, School of Health Sciences, Faculty of Medicine, Kyoto University, Kyoto, Japan, 3Department of Anatomy I, Osaka Medical College, Takatsuki, Japan, 4Department of Pathology, Mie University Graduate School of Medicine, Tsu, Japan, 5Department of Nephrology and Cardiology, International Medical Center of Japan, Tokyo, Japan Histopathological examinations elucidate qualitative and quantitative structural alteration, and detect localization or expression of target substances in tissues or cells. Necrosis, apoptosis, autophagy, and abnormality in the ubiquitin-proteasome system have been proposed as the mechanisms of myocardial cell death or cell degeneration in heart failure. Moreover, inflammatory processes and extracellular matrix remodeling are important factors in the pathophysiology of heart failure. In this presentation, histological, immunohistochemical and electron-microscopic techniques for detecting and estimating these pathological conditions will be reviewed and discussed.
S5-5 Peptidome Analysis of Cardiovascular Tissue and Cells NAOTO MINAMINO, KAZUKI SASAKI Department of Pharmacology, National Cardiovascular Center Research Institute, Suita, Japan Peptides play crucial roles in many physiological events as hormones and local mediators, but the real status of peptides has not been well elucidated. This is mainly due to the facts that most peptides in the cells are degradation products of proteins, and that peptides are present in extremely low concentrations and easily susceptible to proteolysis. Recent progress in the separation and structural analysis by mass spectrometers made it possible to identify a whole set of peptides, PEPTIDOME, in the target. We started the Peptidome project in 1999 and registered peptides of the brain tissue to the Peptidome database. The quality of Peptidome data depends on that of the peptide preparation used for the analysis. However, the peptide quality obtained from cardiovascular tissue is not high in contrast with that from the endocrine tissue. We have been improving the procedures for harvesting peptides from the cardiovascular cells, and very recently succeeded in collecting relatively high quality data including known bioactive peptides. Although more trials are required to optimize the peptide quality, detailed catalogs of the peptides secreted from the cardiovascular cells will be available near in future, which allows us to develop new strategy against the cardiovascular disease.
S5-3 Noncoding RNAs, Emerging Roles in the Regulation of Cardiac Gene Expression MASASHI ARAI, ATAI WATANABE, MASAHIKO KURABAYASHI Gunma University Graduate School of Medicine, Maebashi, Japan Recently, the role of noncoding RNAs, especially small interference RNA (siRNA) and microRNA (miRNA) has been recognized. Double strand RNAs that are transcribed from genome or exogenously administered were processed by slicer enzymes, Drosha and Dicer to form 21w23 nt small RNAs. In the RNA-induced silencing complex, RISC, siRNA cut trget mRNA and miRNA suppresses the translation from target mRNA. SiRNA is especillay useful to suppress specific mRNA expression. We have demonstrated that phospholamban siRNA decreased its mRNA and protein levels to 10% of control 12 hours and 48 hours after transduction, respectively in the rat cardiac myocytes. As a result, Ca2þ uptake rate was increased by 23% estimated by its EC50 value. On the other hand, miRNA is expressed in a spacio-temporal specific manner and suppresses translation from several hundred mRNAs. miR1 decreases Hand2 protein to switch ventricular myocytes from proliferation to differentiation in the developing embryo. miR-195 is increased in the rat pressure overload cardiac hypertrophy and in human DCM heart. Overexpression of miR195 is sufficient to induce cardiac hypertrophy. Accordingly, noncoding RNAs are critically involved in the cardiac development and pathological conditions, and may serve as therapeutic targets in heart disease.
S5-6 Use of DNA Microarray for Cardiovascular Basic Research MASANORI ASAKURA1, HIROSHI ASANUMA2, JIYOONG KIM3, KAZUO KOMAMURA4, HITONOBU TOMOIKE5, MASAFUMI KITAKAZE1 1 Department of R and D of Clinical Research, National Cardiovascular Center, Suita, Japan, 2Kinki University, Osaka-Sayama, Japan, 3National Cardiovascular Center, Cardiovascular Division, Suita, Japan, 4National Cardiovascular Center Research Institute, Suita, Japan, 5National Cardiovascular Center, Suita, Japan Heart failure is one of most critical issues in cardiovascular field all over the world. Cardiologists can use several commercial drugs such as atrial natriuretic peptide, angiotensin II receptor antagonist, beta receptor blocker for the treatment of heart failure. However, most cardiologists think that current therapy for heart failure is not sufficient. It is hoped that new therapy for heart failure could be developed and be applied for the patients with heart failure. To attain this aim, it is important to explore novel targets for the treatment of heart failure. There are several approaches to search for the substances related to the pathogenesis of heart failure. These approaches include transcriptome and proteome. We have analyzed gene expression profiling of heart diseases using DNA microarray. We will present how to use DNA micorarray for the basic research on cardiovascular field, especially on heart failure.