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Increased Protein Phosphatase 1 Activity as a New Therapeutic Target of Heart Failure in Genetic Cardiomyopathy
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Journal of Cardiac Failure Vol. 11 No. 9 Suppl. 2005
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S3-5
Molecular Etiology of Hypertrophic Cardiomyopathy and Dilated Cardiomyopathy; Stiff Sarcomere versus Loose Sarcomere AKINORI KIMURA Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Increased Protein Phosphatase 1 Activity as a New Therapeutic Target of Heart Failure in Genetic Cardiomyopathy YASUHIRO IKEDA, MICHIO YAMADA, KOICHI YOSHIMURA, HIROKI AOKI, MASAFUMI YANO, MASUNORI MATSUZAKI Dept. of Molecular Cardiovascular Biology & Cardiovascular Medicine, Yamaguchi University School of Medicine, Ube Japan
Recent genetic analyses have revealed that mutations in genes for sarcomeric proteins and/or Z-disc components can be found in patients with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), ie, both HCM and DCM can be caused by mutations in different genes and HCM and DCM are allelic diseases. Since HCM and DCM are different in clinical and pathological aspects, it should be clarified why and how the mutations in the same gene cause clinically different diseases. We have recently reported that an HCM-associated mutation in the Z-disc region of titin/connectin (TTN) increases binding to actinin whereas DCM-associated TTN mutations decrease binding to actinin or Tcap/telethonin (TCAP). In addition, HCM-associated TCAP mutations increased binding to titin/ connectin and calsarcin, while DCM-associated TCAP mutations decreased binding to them. These observations suggest that HCM-associated mutations would lead to stiff sarcomere, while DCM-associated mutations lead to loose sarcomere. Stiff and loose sarcomere would increase and decrease passive tension, respectively, resulting in increased and decreased calcium sensitivity of muscle contraction. In addition, we also investigated functional alterations caused by HCM-associated and DCM-associated TTN mutations in the heart-specific N2-B domain, which showed increased and decreased binding to FHL2, respectively. Furthermore, an FHL2 mutation decreasing binding to titin/connectin was found in DCM, further supporting that the allelism was based on the different functional properties caused by the mutations.
Background: The type 1 protein phosphatase (PP1) has been reported to be overactivated in experimental and human failing hearts, leading to a depression of Ca2⫹ cycling and contractility. We previously reported that increased PP1 activity was also observed in the models of genetic cardiomyopathy, UMX cardiomyopathic(CM) hamster, and in vivo inhibition of PP1 by overexpressing inhibitor-2(I-2) improved short-term cardiac function. Method and Results: We here investigated the effect of chronic PP1 inhibition by using high efficiency adeno-associated virus (rAAV-) mediated cardiac I-2 gene delivery on heart failure progression and survival in CM hamster. Endogeonus inhibitor-2 (I-2) was only detected in the microsomal fraction of cardiomyocytes, and adenoviral overexpresion of I-2 preferentially localized in the sarcolemma, suggesting that I-2 is a membrane-bound (m-) endogenous PP1 inhibitor. In addition, PP1 activity following adenovirus I-2 transfection in cardiomyocytes dramatically decreased PP1 activity and augmented Ca2⫹ cycling and cell shortening in cardiomyocytes. rAAV-mediated in vivo cardiac I-2 gene delivery restored cardiac function and extended survival time for 3 months in CM hamsters. Conclusions: These findings suggest that increased m- PP1 in cardiomyocytes is important contributor of depressed Ca2⫹ cycling, and inhibition of m- PP1 via inhibitor-2 may provide a new molecular target for the treatment of heart failure.
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S3-6
How to Apply Genomic Science for the Developments of New Treatment of Chronic Heart Failure MASAFUMI KITAKAZE Cardiovascular Division of Medicine, National Cardiovascular Center, Osaka, JAPAN
Roles of Mitogen-activated Protein Kinase Signaling Pathway in Cardiac Remodeling KINYA OTSU Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine
To find the new pathophysiological features of chronic heart failure (CHF), we applied genomic analyses in patients with CHF. In human failing hearts, DNA array revealed that about 200 and 100 genes are up-regulated and down-regulated among 10,000 genes, respectively. We linked the DNA array database to the clinical parameters of CHF such as ejection fraction, and found several up-regulated genes including HB-EGF or down-regulated genes including adenosine receptors. Either inhibition of HB-EGF or stimulation of adenosine receptors attenuated both hypertrophic signals and cardiac hypertrophy in murine aorta banding(TAC) models. Furthermore, we recognized that the genes responsible for endoplasmic reticulum (ER) stress that causes apoptosis is modulated in patients with CHF. We found that ER chaperones increase after TAC along with the increased TUNEL-positive cells. Among ER-mediated apoptotic pathways, the CHOP-dependent pathway was activated in failing hearts, and RNAi for CHOP prevented the apoptosis. Taken together, HB-EGF, adenosine or ER stress, a new members for the pathophysiology of CHF, may largely contribute to severity of CHF.
Left ventricular remodeling that occurs after myocardial infarction (MI) and pressure overload is generally accepted as a determinant of the clinical course of heart failure. Mitogen activated protein kinases (MAPK) such as ERK, JNK and p38 appear to be involved in this process. We have attempted to elucidate their roles in the pathogenesis of LV remodeling by analyzing the gene-manipulated mice. Apoptosis signal-regulating kinase 1 (ASK1) is a reactive oxygen species-dependent MAPKKK that plays an important role in stress-induced apoptosis. The LV remodeling after coronary artery ligation or thoracic transverse aortic constriction (TAC), manifested as LV dilatation and impaired contractility, was prevented in ASK1 knockout mouse hearts. On the other hands, cardiac-specific overexpression of constitutively active mutant of ASK1 resulted in enhancement of the cardiomyopathic phenotypes. Cardiac-specific p38α knockout mice developed cardiac dysfunction and heart dilatation after TAC. In vitro data indicate that ASK1 in involved only in apoptosis but also in non-apoptotic cell death. Then, in order to examine a clinical relevance of role of ASK1 in LV remodeling, we delivered a dominant-negative mutant of ASK1 in recombinant adeno-associated virus vectors into BIO TO-2 cardiomyopathic hamster model of progressive heart failure using a transcoronary in vivo gene-delivery system. The inhibition of ASK1 activation promoted the improvement of progressive LV remodeling. Taken together, ASK1JNK is involved in cell death signaling pathway, whereas p38 plays a critical role for cardiomyocyte survival pathway. ASK1 will be a valid target for the development of novel therapeutic agents to suppress the progression of heart failure.
Journal of Cardiac Failure Vol. 11 No. 9 Suppl. 2005
S3-3
S3-5
Molecular Etiology of Hypertrophic Cardiomyopathy and Dilated Cardiomyopathy; Stiff Sarcomere versus Loose Sarcomere AKINORI KIMURA Department of Molecular Pathogenesis, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Increased Protein Phosphatase 1 Activity as a New Therapeutic Target of Heart Failure in Genetic Cardiomyopathy YASUHIRO IKEDA, MICHIO YAMADA, KOICHI YOSHIMURA, HIROKI AOKI, MASAFUMI YANO, MASUNORI MATSUZAKI Dept. of Molecular Cardiovascular Biology & Cardiovascular Medicine, Yamaguchi University School of Medicine, Ube Japan
Recent genetic analyses have revealed that mutations in genes for sarcomeric proteins and/or Z-disc components can be found in patients with hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM), ie, both HCM and DCM can be caused by mutations in different genes and HCM and DCM are allelic diseases. Since HCM and DCM are different in clinical and pathological aspects, it should be clarified why and how the mutations in the same gene cause clinically different diseases. We have recently reported that an HCM-associated mutation in the Z-disc region of titin/connectin (TTN) increases binding to actinin whereas DCM-associated TTN mutations decrease binding to actinin or Tcap/telethonin (TCAP). In addition, HCM-associated TCAP mutations increased binding to titin/ connectin and calsarcin, while DCM-associated TCAP mutations decreased binding to them. These observations suggest that HCM-associated mutations would lead to stiff sarcomere, while DCM-associated mutations lead to loose sarcomere. Stiff and loose sarcomere would increase and decrease passive tension, respectively, resulting in increased and decreased calcium sensitivity of muscle contraction. In addition, we also investigated functional alterations caused by HCM-associated and DCM-associated TTN mutations in the heart-specific N2-B domain, which showed increased and decreased binding to FHL2, respectively. Furthermore, an FHL2 mutation decreasing binding to titin/connectin was found in DCM, further supporting that the allelism was based on the different functional properties caused by the mutations.
Background: The type 1 protein phosphatase (PP1) has been reported to be overactivated in experimental and human failing hearts, leading to a depression of Ca2⫹ cycling and contractility. We previously reported that increased PP1 activity was also observed in the models of genetic cardiomyopathy, UMX cardiomyopathic(CM) hamster, and in vivo inhibition of PP1 by overexpressing inhibitor-2(I-2) improved short-term cardiac function. Method and Results: We here investigated the effect of chronic PP1 inhibition by using high efficiency adeno-associated virus (rAAV-) mediated cardiac I-2 gene delivery on heart failure progression and survival in CM hamster. Endogeonus inhibitor-2 (I-2) was only detected in the microsomal fraction of cardiomyocytes, and adenoviral overexpresion of I-2 preferentially localized in the sarcolemma, suggesting that I-2 is a membrane-bound (m-) endogenous PP1 inhibitor. In addition, PP1 activity following adenovirus I-2 transfection in cardiomyocytes dramatically decreased PP1 activity and augmented Ca2⫹ cycling and cell shortening in cardiomyocytes. rAAV-mediated in vivo cardiac I-2 gene delivery restored cardiac function and extended survival time for 3 months in CM hamsters. Conclusions: These findings suggest that increased m- PP1 in cardiomyocytes is important contributor of depressed Ca2⫹ cycling, and inhibition of m- PP1 via inhibitor-2 may provide a new molecular target for the treatment of heart failure.
S3-4
S3-6
How to Apply Genomic Science for the Developments of New Treatment of Chronic Heart Failure MASAFUMI KITAKAZE Cardiovascular Division of Medicine, National Cardiovascular Center, Osaka, JAPAN
Roles of Mitogen-activated Protein Kinase Signaling Pathway in Cardiac Remodeling KINYA OTSU Department of Cardiovascular Medicine, Osaka University Graduate School of Medicine
To find the new pathophysiological features of chronic heart failure (CHF), we applied genomic analyses in patients with CHF. In human failing hearts, DNA array revealed that about 200 and 100 genes are up-regulated and down-regulated among 10,000 genes, respectively. We linked the DNA array database to the clinical parameters of CHF such as ejection fraction, and found several up-regulated genes including HB-EGF or down-regulated genes including adenosine receptors. Either inhibition of HB-EGF or stimulation of adenosine receptors attenuated both hypertrophic signals and cardiac hypertrophy in murine aorta banding(TAC) models. Furthermore, we recognized that the genes responsible for endoplasmic reticulum (ER) stress that causes apoptosis is modulated in patients with CHF. We found that ER chaperones increase after TAC along with the increased TUNEL-positive cells. Among ER-mediated apoptotic pathways, the CHOP-dependent pathway was activated in failing hearts, and RNAi for CHOP prevented the apoptosis. Taken together, HB-EGF, adenosine or ER stress, a new members for the pathophysiology of CHF, may largely contribute to severity of CHF.
Left ventricular remodeling that occurs after myocardial infarction (MI) and pressure overload is generally accepted as a determinant of the clinical course of heart failure. Mitogen activated protein kinases (MAPK) such as ERK, JNK and p38 appear to be involved in this process. We have attempted to elucidate their roles in the pathogenesis of LV remodeling by analyzing the gene-manipulated mice. Apoptosis signal-regulating kinase 1 (ASK1) is a reactive oxygen species-dependent MAPKKK that plays an important role in stress-induced apoptosis. The LV remodeling after coronary artery ligation or thoracic transverse aortic constriction (TAC), manifested as LV dilatation and impaired contractility, was prevented in ASK1 knockout mouse hearts. On the other hands, cardiac-specific overexpression of constitutively active mutant of ASK1 resulted in enhancement of the cardiomyopathic phenotypes. Cardiac-specific p38α knockout mice developed cardiac dysfunction and heart dilatation after TAC. In vitro data indicate that ASK1 in involved only in apoptosis but also in non-apoptotic cell death. Then, in order to examine a clinical relevance of role of ASK1 in LV remodeling, we delivered a dominant-negative mutant of ASK1 in recombinant adeno-associated virus vectors into BIO TO-2 cardiomyopathic hamster model of progressive heart failure using a transcoronary in vivo gene-delivery system. The inhibition of ASK1 activation promoted the improvement of progressive LV remodeling. Taken together, ASK1JNK is involved in cell death signaling pathway, whereas p38 plays a critical role for cardiomyocyte survival pathway. ASK1 will be a valid target for the development of novel therapeutic agents to suppress the progression of heart failure.