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Adenoviral-Mediated Murine Tissue Inhibitor of Metalloproteinse-2 Gene Transfer Confers Survival Benefits in a Murine Model of Myocardial Infarction
The 9th Annual Scientific Meeting
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HFSA
S127
134
136
TRPC Channels and b1-Adrenergic Mediated Activation of the Fetal Gene Program Carmen C. Sucharov1, Michael R. Bristow1; 1Cardiology, UCHSC, Denver, CO
Adenoviral-Mediated Murine Tissue Inhibitor of Metalloproteinse-2 Gene Transfer Confers Survival Benefits in a Murine Model of Myocardial Infarction Ravi Ramani1, Ish Singla1, Kathy Nilles1, Benjamin Burkhead1, Jennifer Dawson1, Gregory Gibson1, Charles McTiernan1; 1Cardiovascular Institute, University of Pittsburgh Medical Center, Pittsburgh, PA
Transient receptor potential channels (TRPC) constitute a family of channels that have been proposed to function as store-operated or second messenger-operated channels in a variety of cell types. Recently, TRPC3 has been implicated in the calcineurin-mediated response in skeletal muscle, making it a likely candidate for mediating hypertrophy in the heart. Activation of the fetal gene program during pathologic hypertrophy is characterized by changes in gene expression that includes repression of genes that are expressed during adult development (a-Myosin Heavy Chain (a-MyHC) and sarcoplasmatic reticulum ATPase 2a (SRCA2a)), and re-expression of genes that are present during the fetal development (b-Myosin Heavy Chain (b-MyHC), atrial and brain natriuretic peptide (ANP and BNP), and skeletal a-actin). We have recently shown that dilated cardiomyopathy patients (DCM) patients have changes in gene expression that recapitulate the fetal gene program, and that these changes are partially reversed in patients that favorably respond to b-blocker therapy. We have also shown that b1adernegic stimulation in neonatal rat cardiac myocytes results in changes in the fetal gene program consistent with pathologic hypertrophic response. Here we show that TRPC1 mRNA levels and TRPC5 mRNA levels are up-regulated 6 fold and 8 fold respectively in human heart failure. Moreover, we show that TRPC blockade in neonatal rat cardiac myocytes prevents the activation of the fetal gene program in response to b1-adrenergic stimulation. These results suggest that TRPCs are an important component of the pathways involved in the induction of fetal gene program during pathologic hypertrophy.
Background: Cardiac extracellular matrix homeostasis is mediated primarily through a balance between the degradative matrix metalloproteinases (MMPs) and their specific inhibitors called tissue inhibitors of metalloproteinases (TIMPs 1-4). Hypothesis: Correction of post myocardial infarction (MI) MMP-TIMP imbalances through adenoviral-mediated gene delivery of TIMP-2 (AdV-T2) would ameliorate ventricular remodeling and improve survival in a murine model of MI. Methods: Male mice (n ⫽ 49) aged 10–14 weeks underwent either left coronary artery ligation to induce myocardial infarction (MI group) (n ⫽ 26), or received myocardial injection of 6*1010 viral particles of AdV-T2 immediately post-MI(T2⫹MI group)(n ⫽ 18), or were controls (n ⫽ 5).At days 3 and 7 surviving mice underwent echocardiography for assessment of left ventricular (LV) size and function, and were sacrificed. Excised hearts were weighed, and the LV analyzed for MMP-2 and -9, and TIMP-1 and -2 protein levels using activity assays and ELISAs respectively. Results: 24 mice survived till the end of the 7 day study period. Survival was significantly better in the T2⫹MI group infarcted animals treated with TIMP compared with the MI group (55 % vs. 34 % respectively, p ⬍ 0.02; see figure). The major cause of death was cardiac rupture. There were no significant differences between infarcted groups in LV weight, size or function. MMP2 and -9, and TIMP-1 levels were higher in infarcted hearts compared to controls, but there was no significant difference between MI and T2⫹MI groups. TIMP-2 levels however were highest in the T2⫹MI group (7.9 ⫾ 2.2, 4.6 ⫾ 1.9, and 0.94 ⫾ 0.12 ng/100 µG protein in T2⫹MI, MI and control groups respectively, p ⬍ 0.02). Conclusions: Adenoviral mediated myocardial delivery of the TIMP-2 gene protects against cardiac rupture in a murine model of myocardial infarction, but did not protect against adverse remodeling. Survival benefit correlates with a partial restoration of MMP-TIMP ratios. Further studies are needed to determine the mechanism of this protective effect.
135 Regulation of NF-kB Gene Transcription by Class I HDACs in Ventricular Myocytes Delphine Baetz1, James Shaw1, Kelly M. Regula1, Tong Zhang1, Lorrie A. Kirshenbaum1; 1Institute of Cardiovascular Sciences, University of Manitoba, Winnipeg, MB, Canada Programmed cell death or apoptosis has been recognized to play a central role in a number of cardiovascular pathologies including heart failure. Transcription factor NFkB is known to activate a survival pathway in mammalian cells. In this report we show that the transcription potential of the p65 subunit of NFkB is regulated by the class I histone deacetylase proteins (HDAC). We demonstrate that expression of the p65 subunit of NF-kB resulted in a 10 fold increase in NF-kB dependent transcription in post-natal ventricular myocytes that was repressed to basal level by HDAC 1 and HDAC 2 activity. Inhibition of HDAC activity with trichostatin A (100 mM TSA) abrogated the inhibitory effects of HDAC on NFkB dependent gene activation. Mutations in the catalytic domains of HDAC 1 (H141A), crucial for de-acetylation, derepressed the inhibitory effects of HDAC on NF-kB gene activation. Immunoprecipitation assays revealed that HDAC1 but not HDAC 2 formed protein-protein interactions with p65 subunit of NF-kB. Interestingly, 2.5 fold reduction in HDAC activity was observed in post-natal ventricular myocytes subjected to hypoxia. To our knowledge the data provide the first evidence that survival pathways mediated by NF-kB are regulated by class I HDACs in post-natal ventricular myocytes.
137 Friend of GATA-2 (FOG-2) Physically Binds and Represses Thyroid Hormone Receptor-Dependent Regulation of the SERCA2 Gene Promoter Rosanne Rouf1, Jing Wu1, Jay Boltax1, Gordon S. Huggins1; 1Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, MA Reduced expression of sarcoplasmic reticulum calcium ATPase-2 (SERCA2) contributes directly to the reduced mechanical efficiency of the failing heart. SERCA2 expression is regulated by thyroid hormone, and reduced SERCA2 expression in the euthyroid failing heart is considered to reflect an intrinsic defect of thyroid hormone receptor-dependent gene expression. Although several mechanisms of thyroid hormone resistance in the cardiomyocyte have been proposed we considered whether the Friend of GATA-2 (FOG-2) cardiac co-repressor protein, which is necessary for heart formation and regulation of fetal gene expression, would block SERCA2 expression selectively in the failing heart. In support of this hypothesis we found that human heart samples obtained at the time of left ventricular assist device placement and mouse heart from a cardiomyopathy model show increased FOG-2 expression compared with control tissue. Previously we have shown that FOG-2 selectively targets the COUP-TF orphan nuclear hormone receptors. Using co-immunoprecipitation and GST pull-down assays we have found that FOG-2 also physically binds the fulllength thyroid hormone receptor 1-alpha (TRa1) isoform in the presence and the absence of thyroid hormone. This interaction was specific because the seventh and eighth FOG-2 zinc fingers bind TRa1, yet not the TRa2 isoform which has a distinct ligand-binding domain. Furthermore this interaction is important because co-expression of FOG-2 significantly blocked TRa1-dependent thyroid hormone-stimulated gene expression in CV-1 cells and rat neonatal cardiomyocytes. To determine if increased FOG-2 expression alone could alter gene expression in the adult heart we overexpressed FOG-2 from the myosin heavy chain gene promoter in transgenic mice (MHC-FOG-2). Microarray analysis detected multiple transcript changes consistent with induction of the fetal gene program common to the failing heart. Transcript and Western blotting studies confirmed significant SERCA2 downregulation. Finally, overexpression of FOG-2 lowered the transcriptional activity of thyroid hormone stimulated SERCA2(⫺585 to ⫹420)-luciferase reporter plasmid in cultured chick ventricular cardiomyocytes. These data support our model that FOG-2 may contribute broadly to changes in gene expression seen in the failing heart by blocking thyroid hormone-dependent gene expression.
•
HFSA
S127
134
136
TRPC Channels and b1-Adrenergic Mediated Activation of the Fetal Gene Program Carmen C. Sucharov1, Michael R. Bristow1; 1Cardiology, UCHSC, Denver, CO
Adenoviral-Mediated Murine Tissue Inhibitor of Metalloproteinse-2 Gene Transfer Confers Survival Benefits in a Murine Model of Myocardial Infarction Ravi Ramani1, Ish Singla1, Kathy Nilles1, Benjamin Burkhead1, Jennifer Dawson1, Gregory Gibson1, Charles McTiernan1; 1Cardiovascular Institute, University of Pittsburgh Medical Center, Pittsburgh, PA
Transient receptor potential channels (TRPC) constitute a family of channels that have been proposed to function as store-operated or second messenger-operated channels in a variety of cell types. Recently, TRPC3 has been implicated in the calcineurin-mediated response in skeletal muscle, making it a likely candidate for mediating hypertrophy in the heart. Activation of the fetal gene program during pathologic hypertrophy is characterized by changes in gene expression that includes repression of genes that are expressed during adult development (a-Myosin Heavy Chain (a-MyHC) and sarcoplasmatic reticulum ATPase 2a (SRCA2a)), and re-expression of genes that are present during the fetal development (b-Myosin Heavy Chain (b-MyHC), atrial and brain natriuretic peptide (ANP and BNP), and skeletal a-actin). We have recently shown that dilated cardiomyopathy patients (DCM) patients have changes in gene expression that recapitulate the fetal gene program, and that these changes are partially reversed in patients that favorably respond to b-blocker therapy. We have also shown that b1adernegic stimulation in neonatal rat cardiac myocytes results in changes in the fetal gene program consistent with pathologic hypertrophic response. Here we show that TRPC1 mRNA levels and TRPC5 mRNA levels are up-regulated 6 fold and 8 fold respectively in human heart failure. Moreover, we show that TRPC blockade in neonatal rat cardiac myocytes prevents the activation of the fetal gene program in response to b1-adrenergic stimulation. These results suggest that TRPCs are an important component of the pathways involved in the induction of fetal gene program during pathologic hypertrophy.
Background: Cardiac extracellular matrix homeostasis is mediated primarily through a balance between the degradative matrix metalloproteinases (MMPs) and their specific inhibitors called tissue inhibitors of metalloproteinases (TIMPs 1-4). Hypothesis: Correction of post myocardial infarction (MI) MMP-TIMP imbalances through adenoviral-mediated gene delivery of TIMP-2 (AdV-T2) would ameliorate ventricular remodeling and improve survival in a murine model of MI. Methods: Male mice (n ⫽ 49) aged 10–14 weeks underwent either left coronary artery ligation to induce myocardial infarction (MI group) (n ⫽ 26), or received myocardial injection of 6*1010 viral particles of AdV-T2 immediately post-MI(T2⫹MI group)(n ⫽ 18), or were controls (n ⫽ 5).At days 3 and 7 surviving mice underwent echocardiography for assessment of left ventricular (LV) size and function, and were sacrificed. Excised hearts were weighed, and the LV analyzed for MMP-2 and -9, and TIMP-1 and -2 protein levels using activity assays and ELISAs respectively. Results: 24 mice survived till the end of the 7 day study period. Survival was significantly better in the T2⫹MI group infarcted animals treated with TIMP compared with the MI group (55 % vs. 34 % respectively, p ⬍ 0.02; see figure). The major cause of death was cardiac rupture. There were no significant differences between infarcted groups in LV weight, size or function. MMP2 and -9, and TIMP-1 levels were higher in infarcted hearts compared to controls, but there was no significant difference between MI and T2⫹MI groups. TIMP-2 levels however were highest in the T2⫹MI group (7.9 ⫾ 2.2, 4.6 ⫾ 1.9, and 0.94 ⫾ 0.12 ng/100 µG protein in T2⫹MI, MI and control groups respectively, p ⬍ 0.02). Conclusions: Adenoviral mediated myocardial delivery of the TIMP-2 gene protects against cardiac rupture in a murine model of myocardial infarction, but did not protect against adverse remodeling. Survival benefit correlates with a partial restoration of MMP-TIMP ratios. Further studies are needed to determine the mechanism of this protective effect.
135 Regulation of NF-kB Gene Transcription by Class I HDACs in Ventricular Myocytes Delphine Baetz1, James Shaw1, Kelly M. Regula1, Tong Zhang1, Lorrie A. Kirshenbaum1; 1Institute of Cardiovascular Sciences, University of Manitoba, Winnipeg, MB, Canada Programmed cell death or apoptosis has been recognized to play a central role in a number of cardiovascular pathologies including heart failure. Transcription factor NFkB is known to activate a survival pathway in mammalian cells. In this report we show that the transcription potential of the p65 subunit of NFkB is regulated by the class I histone deacetylase proteins (HDAC). We demonstrate that expression of the p65 subunit of NF-kB resulted in a 10 fold increase in NF-kB dependent transcription in post-natal ventricular myocytes that was repressed to basal level by HDAC 1 and HDAC 2 activity. Inhibition of HDAC activity with trichostatin A (100 mM TSA) abrogated the inhibitory effects of HDAC on NFkB dependent gene activation. Mutations in the catalytic domains of HDAC 1 (H141A), crucial for de-acetylation, derepressed the inhibitory effects of HDAC on NF-kB gene activation. Immunoprecipitation assays revealed that HDAC1 but not HDAC 2 formed protein-protein interactions with p65 subunit of NF-kB. Interestingly, 2.5 fold reduction in HDAC activity was observed in post-natal ventricular myocytes subjected to hypoxia. To our knowledge the data provide the first evidence that survival pathways mediated by NF-kB are regulated by class I HDACs in post-natal ventricular myocytes.
137 Friend of GATA-2 (FOG-2) Physically Binds and Represses Thyroid Hormone Receptor-Dependent Regulation of the SERCA2 Gene Promoter Rosanne Rouf1, Jing Wu1, Jay Boltax1, Gordon S. Huggins1; 1Molecular Cardiology Research Institute, Tufts-New England Medical Center, Boston, MA Reduced expression of sarcoplasmic reticulum calcium ATPase-2 (SERCA2) contributes directly to the reduced mechanical efficiency of the failing heart. SERCA2 expression is regulated by thyroid hormone, and reduced SERCA2 expression in the euthyroid failing heart is considered to reflect an intrinsic defect of thyroid hormone receptor-dependent gene expression. Although several mechanisms of thyroid hormone resistance in the cardiomyocyte have been proposed we considered whether the Friend of GATA-2 (FOG-2) cardiac co-repressor protein, which is necessary for heart formation and regulation of fetal gene expression, would block SERCA2 expression selectively in the failing heart. In support of this hypothesis we found that human heart samples obtained at the time of left ventricular assist device placement and mouse heart from a cardiomyopathy model show increased FOG-2 expression compared with control tissue. Previously we have shown that FOG-2 selectively targets the COUP-TF orphan nuclear hormone receptors. Using co-immunoprecipitation and GST pull-down assays we have found that FOG-2 also physically binds the fulllength thyroid hormone receptor 1-alpha (TRa1) isoform in the presence and the absence of thyroid hormone. This interaction was specific because the seventh and eighth FOG-2 zinc fingers bind TRa1, yet not the TRa2 isoform which has a distinct ligand-binding domain. Furthermore this interaction is important because co-expression of FOG-2 significantly blocked TRa1-dependent thyroid hormone-stimulated gene expression in CV-1 cells and rat neonatal cardiomyocytes. To determine if increased FOG-2 expression alone could alter gene expression in the adult heart we overexpressed FOG-2 from the myosin heavy chain gene promoter in transgenic mice (MHC-FOG-2). Microarray analysis detected multiple transcript changes consistent with induction of the fetal gene program common to the failing heart. Transcript and Western blotting studies confirmed significant SERCA2 downregulation. Finally, overexpression of FOG-2 lowered the transcriptional activity of thyroid hormone stimulated SERCA2(⫺585 to ⫹420)-luciferase reporter plasmid in cultured chick ventricular cardiomyocytes. These data support our model that FOG-2 may contribute broadly to changes in gene expression seen in the failing heart by blocking thyroid hormone-dependent gene expression.