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Overexpression of tumor necrosis factor-α activates the expression of multiple members of the apoptosis pathway in transgenic mice
The Second Annual Scientific Meeting
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HFSA
083
084
The Human Phospholamban Gene: Expression and Structure
Overexpression of Tumor Necrosis Faetor-c~Activates the Expression of Multiple Members of the Apoptosis Pathway in Transgenic Mice
Charles F. McTierrtan, Carole Frye, Bonnie Lemster, Erie A. Kinder, Christine Moravee, Arthur M. Feldman, University of Pittsburgh, Pittsburgh PA Phospholamban (PLB) is a key regulator of sarcoplasmic reticulum calcitma uptake and thus cardiac contractility. Variations in the level of PLB expression can contribute to alterations in muscle functional properties, thus understanding regulation ofPLB gene transcription may reveal new approaches to alter cardiac physiology through modification of PLB expression. To better characterize the expression and regulation of PLB in huraanfissues, andto begin identification of regulatory elements of the human PLB gene, we have (1) assessed the expression of PLB transcripts in various human tissues and in normal and diseased human hearts, (2) isolated human PLB genomic sequences and characterized genomic structure, and (3) assessed promoter activity of human PLB reporter constructs. PLB is expressed at high levels in human ventricle and skeletal muscles, with trace levels of expression in various smooth muscle organs. Human atria express cons~iderablylower levels of PLB transcripts than do ventricles. In heart failure the level of ventrioflar PLB transcripts is significantly reduced relative to that of non-failing hearts (non-failing 100% n = 9; falling n=22, 72% p<0.01). Screening of a human genomic library yielded 3 clones containing PLB sequences. The human PLB gene has two exons (0.1 and <3 kb) separated by a single intrun (approximately 9.5 kb) similar to the PLB gene of other species. Primer extension analysis identified the transcription start site at a sequence similar to that reported for rat. Sequence analysis of the proximal promoter regions identifies striking conservation between human and 4 other mammalian PLB genes within the 170 bp upstream of the transcription start site. This includes conserved CP-1 and GATAA boxes, and a novel motif that we previously found to be required for rat PLB promoter activity in transient transfection assays. Reporter constructs containing sequences -169/+64 of the human PLB gene are active when transfected into neonatal rat cardiomyocyntes. Titus the human PLB promoter is structurally related to other mammalian PLB genes, down-regulated in the myocardium during heart failure, expressed at relatively high levels in skeletal muscles, and shows marked conservation of promoter regulatory elements identified in the rat PLB gene.
Toru Kubota, M a s a y u k i Miyagishima, George g. Bounoutas, Charles F.McTiernan, Arthur M. Feldman, University of Pittsburgh, Pittsburgh, PA Tumor necrosis factor (TNF)-c~ is a potent inducer of apoptosis. We have previously reported that transgenic mice with cardiac-specific overexpression of TNF-c~ (TG) develop myocarditis, cardiomegaly, and congestive heart failure. In addition, we observed apoptosis in the myocytes and the interstitial ceils. However, the signaling pathways responsible for TNF-c~ induced apoptosis in this mouse model remain undefined. Therefore, we assessed the expression of a group of genes thought to play a role in the apoptotic pathway. RNA was extracted fi:om left ventricles of 8-week-old TG and wild-type controls (WT, n = 6 each) Multi-probe ribocuclease protection assays were used to evaluate transcripts of apoptosis-related genes. The value of each transcript was normalized to that of GAPDH (= 1). Increased expression of Pus (TG / W T = 0.032? / 0.0110), FasL (0.0021 / 0.0000), FADD (0.0242 / 0.0148), and FLICE (0.0599 / 0.0119) was observed in TG (p<0.01). Other caspases such as ICE (0.0180 / 0.0016), ICH (0.0108 / 0.0041), YAMA (0.0191 / 0.0025), Caspase 11 (0.0195 / 0.0011), Caspase 12 (0.0429 / 0.0040), Mch2 (0.0171 / 0.0082), Mch3 (0.0142 / 0.0054) were also increased (p<0.01). We also evaluated expression of bcl-2 family genes in the heart. Both proapoptotic box (0.0555 / 0.0213), bak (0.0225 / 0.0067), and bad (0.0122 / 0.0059), and antiapoptotic bcl-2 (0.0076 / 0.0029) and bfil (0.2524 / 0.00?6) were induced by TNF-c~ overexpression (p<0.01). Increased incidence of apoptosis in TG was shown by DNA laddering as well as TUN'EL assays. In conclusion, cardiac-specific overexpression of TNF-c~ induces a large number of apoptosis-related genes in the myocardium, resulting in increased apoptosis of Interstitial cells and myocytes.
085
086
Adenoviral-Directed Overexpression of Soluble Tumor Necrosis Factor Receptors Reverses Myocarditis in Transgenie Mice With Congestive Heart Failure
Unregulation and Persistent Activation of c-Jun NH2-Terminal Kinases in the Ischemic Failing Human Heart
George S. Bounoutas, Toru Kubota, Masayuki Miyagishima, Charles F. McTiernan, Christina Bruton, Paul D. Robbins, Arthur M. Feldman, University of Pittsburgh, Pittsburgh, PA Transgenic (TG) mice overexpressing tumor necrosis factor (TNF)-c~ in the myocardium develop interstitial infiltrates, cardiac hypertrophy and dilatation, and systolic dysfunction. The TG hearts demonstrate activation of the fetal gene program with increased expression of atrial natriuretic factor (ANF) and t3-myosinheavy chain (MHC). Furthermore, they demonstrated an induction of other cytokines including interleukin (IL)-1J3, We hypothesized that phenotypic changes in the TG mouse could be abrogated by the overexpression of soluble TNF receptors. To test this hypothesis, TG mice were injected retroorbitally with replication deficient adenoviral vectors which expressed either the human soluble TNF receptor type I lgG fusion protein (hsTNFRI) or LacZ at six weeks of age (n = 6 each). Mice were sacrificed two weeks after treatment and analyzed. An adenoviral dose of I09 pfu resulted in high levels of hsTNFRI in plasma (45-180 gg/ml) and in myocardial tissue (51-92 ng/mg). The number of infil~ating cells in the myocardium was reduced to wild type levels in hsTNFRI treated animals (p < 0.0I), but the ventricle weight to body weight ratio remained unchanged in comparison to transgenic and LacZ treated animals. Expression of IL-1 [3and other cytokines was decreased to near wild type levels in hsTNFRI treated animals (p < 0.05), but the reexpression of ANF and J3MHC was unaffected by treatment with soluble receptors. In conclusion, two-week treatment with hsTNFRI abrogated induction of proinflamatory cytokines and myocarditis, but did not block the re-expression of fetal cardiac genes and hypertrophy process. These results suggest that cardiac remodelling may require longer treatment to be reversed, which may have clinical implications.
23
Yun You Li, Charles F. McTiernan, Arthur M. Feldman. Research Laboratories, Division of Cardiology, University of Pittsburgh, Pittsburgh, PA 15213 We have previously reported that the expression and activity of matrix metalloproteinase 9 are increased in the failing human heart, The activation of c-Jun NH2-terminal kinases (JNKs) has been suggested to be a critical step in the induction of matrix metalloproteinases through the activation of transcription factor activator protein-1. Thus, we hypothesized that JNKs may be activated in the failing human heart. To test this hypothesis, JNKI protein and phosphorylated JNK1/JN~(2 (pJNK1/p-JNK2) proteins were detected by Western analysis using respective specific antibodies, the activities of JNKs measured by immune complex JNK assay in the non-failing (u= 7), dilated cardiomyopathic (DCM, n = 7) and ischemic cardiomyopathic (ICM, n= 8) failing human ventricular myocardium of the same groups of patients we previously studied for the expression and activities of matrix metalloproteinnses. We demonstrated that the JNK1 protein levels and pJNK1/p-JNK2 protein levels were significantly increased in DCM heart (8-fold increase for both p-JNK1 and p-JNK2, P < .05), as well as in ICM heart (30-fold increase for p-JNK1, and 15-fold increase for pJNK2, P < .05) as compared to non-failing controls. The activities of JNKs to phosphorylate GST-c-Jun were increased by 2-fold in ICM failing heart (P < .05), but not in DCM failing heart. These results selectively demonstrated that JNKs were upregulated and persistently activated in the ischemic 5tiling human heart. Given the fact that experimental myocardial ischemia followed by reperfusion induces activation of JNKs, the upregulation and persistent activation of JNKs in the ischemic failing human heart may be a result as well as an indicator of repeated ischemia with reperfusion.
•
HFSA
083
084
The Human Phospholamban Gene: Expression and Structure
Overexpression of Tumor Necrosis Faetor-c~Activates the Expression of Multiple Members of the Apoptosis Pathway in Transgenic Mice
Charles F. McTierrtan, Carole Frye, Bonnie Lemster, Erie A. Kinder, Christine Moravee, Arthur M. Feldman, University of Pittsburgh, Pittsburgh PA Phospholamban (PLB) is a key regulator of sarcoplasmic reticulum calcitma uptake and thus cardiac contractility. Variations in the level of PLB expression can contribute to alterations in muscle functional properties, thus understanding regulation ofPLB gene transcription may reveal new approaches to alter cardiac physiology through modification of PLB expression. To better characterize the expression and regulation of PLB in huraanfissues, andto begin identification of regulatory elements of the human PLB gene, we have (1) assessed the expression of PLB transcripts in various human tissues and in normal and diseased human hearts, (2) isolated human PLB genomic sequences and characterized genomic structure, and (3) assessed promoter activity of human PLB reporter constructs. PLB is expressed at high levels in human ventricle and skeletal muscles, with trace levels of expression in various smooth muscle organs. Human atria express cons~iderablylower levels of PLB transcripts than do ventricles. In heart failure the level of ventrioflar PLB transcripts is significantly reduced relative to that of non-failing hearts (non-failing 100% n = 9; falling n=22, 72% p<0.01). Screening of a human genomic library yielded 3 clones containing PLB sequences. The human PLB gene has two exons (0.1 and <3 kb) separated by a single intrun (approximately 9.5 kb) similar to the PLB gene of other species. Primer extension analysis identified the transcription start site at a sequence similar to that reported for rat. Sequence analysis of the proximal promoter regions identifies striking conservation between human and 4 other mammalian PLB genes within the 170 bp upstream of the transcription start site. This includes conserved CP-1 and GATAA boxes, and a novel motif that we previously found to be required for rat PLB promoter activity in transient transfection assays. Reporter constructs containing sequences -169/+64 of the human PLB gene are active when transfected into neonatal rat cardiomyocyntes. Titus the human PLB promoter is structurally related to other mammalian PLB genes, down-regulated in the myocardium during heart failure, expressed at relatively high levels in skeletal muscles, and shows marked conservation of promoter regulatory elements identified in the rat PLB gene.
Toru Kubota, M a s a y u k i Miyagishima, George g. Bounoutas, Charles F.McTiernan, Arthur M. Feldman, University of Pittsburgh, Pittsburgh, PA Tumor necrosis factor (TNF)-c~ is a potent inducer of apoptosis. We have previously reported that transgenic mice with cardiac-specific overexpression of TNF-c~ (TG) develop myocarditis, cardiomegaly, and congestive heart failure. In addition, we observed apoptosis in the myocytes and the interstitial ceils. However, the signaling pathways responsible for TNF-c~ induced apoptosis in this mouse model remain undefined. Therefore, we assessed the expression of a group of genes thought to play a role in the apoptotic pathway. RNA was extracted fi:om left ventricles of 8-week-old TG and wild-type controls (WT, n = 6 each) Multi-probe ribocuclease protection assays were used to evaluate transcripts of apoptosis-related genes. The value of each transcript was normalized to that of GAPDH (= 1). Increased expression of Pus (TG / W T = 0.032? / 0.0110), FasL (0.0021 / 0.0000), FADD (0.0242 / 0.0148), and FLICE (0.0599 / 0.0119) was observed in TG (p<0.01). Other caspases such as ICE (0.0180 / 0.0016), ICH (0.0108 / 0.0041), YAMA (0.0191 / 0.0025), Caspase 11 (0.0195 / 0.0011), Caspase 12 (0.0429 / 0.0040), Mch2 (0.0171 / 0.0082), Mch3 (0.0142 / 0.0054) were also increased (p<0.01). We also evaluated expression of bcl-2 family genes in the heart. Both proapoptotic box (0.0555 / 0.0213), bak (0.0225 / 0.0067), and bad (0.0122 / 0.0059), and antiapoptotic bcl-2 (0.0076 / 0.0029) and bfil (0.2524 / 0.00?6) were induced by TNF-c~ overexpression (p<0.01). Increased incidence of apoptosis in TG was shown by DNA laddering as well as TUN'EL assays. In conclusion, cardiac-specific overexpression of TNF-c~ induces a large number of apoptosis-related genes in the myocardium, resulting in increased apoptosis of Interstitial cells and myocytes.
085
086
Adenoviral-Directed Overexpression of Soluble Tumor Necrosis Factor Receptors Reverses Myocarditis in Transgenie Mice With Congestive Heart Failure
Unregulation and Persistent Activation of c-Jun NH2-Terminal Kinases in the Ischemic Failing Human Heart
George S. Bounoutas, Toru Kubota, Masayuki Miyagishima, Charles F. McTiernan, Christina Bruton, Paul D. Robbins, Arthur M. Feldman, University of Pittsburgh, Pittsburgh, PA Transgenic (TG) mice overexpressing tumor necrosis factor (TNF)-c~ in the myocardium develop interstitial infiltrates, cardiac hypertrophy and dilatation, and systolic dysfunction. The TG hearts demonstrate activation of the fetal gene program with increased expression of atrial natriuretic factor (ANF) and t3-myosinheavy chain (MHC). Furthermore, they demonstrated an induction of other cytokines including interleukin (IL)-1J3, We hypothesized that phenotypic changes in the TG mouse could be abrogated by the overexpression of soluble TNF receptors. To test this hypothesis, TG mice were injected retroorbitally with replication deficient adenoviral vectors which expressed either the human soluble TNF receptor type I lgG fusion protein (hsTNFRI) or LacZ at six weeks of age (n = 6 each). Mice were sacrificed two weeks after treatment and analyzed. An adenoviral dose of I09 pfu resulted in high levels of hsTNFRI in plasma (45-180 gg/ml) and in myocardial tissue (51-92 ng/mg). The number of infil~ating cells in the myocardium was reduced to wild type levels in hsTNFRI treated animals (p < 0.0I), but the ventricle weight to body weight ratio remained unchanged in comparison to transgenic and LacZ treated animals. Expression of IL-1 [3and other cytokines was decreased to near wild type levels in hsTNFRI treated animals (p < 0.05), but the reexpression of ANF and J3MHC was unaffected by treatment with soluble receptors. In conclusion, two-week treatment with hsTNFRI abrogated induction of proinflamatory cytokines and myocarditis, but did not block the re-expression of fetal cardiac genes and hypertrophy process. These results suggest that cardiac remodelling may require longer treatment to be reversed, which may have clinical implications.
23
Yun You Li, Charles F. McTiernan, Arthur M. Feldman. Research Laboratories, Division of Cardiology, University of Pittsburgh, Pittsburgh, PA 15213 We have previously reported that the expression and activity of matrix metalloproteinase 9 are increased in the failing human heart, The activation of c-Jun NH2-terminal kinases (JNKs) has been suggested to be a critical step in the induction of matrix metalloproteinases through the activation of transcription factor activator protein-1. Thus, we hypothesized that JNKs may be activated in the failing human heart. To test this hypothesis, JNKI protein and phosphorylated JNK1/JN~(2 (pJNK1/p-JNK2) proteins were detected by Western analysis using respective specific antibodies, the activities of JNKs measured by immune complex JNK assay in the non-failing (u= 7), dilated cardiomyopathic (DCM, n = 7) and ischemic cardiomyopathic (ICM, n= 8) failing human ventricular myocardium of the same groups of patients we previously studied for the expression and activities of matrix metalloproteinnses. We demonstrated that the JNK1 protein levels and pJNK1/p-JNK2 protein levels were significantly increased in DCM heart (8-fold increase for both p-JNK1 and p-JNK2, P < .05), as well as in ICM heart (30-fold increase for p-JNK1, and 15-fold increase for pJNK2, P < .05) as compared to non-failing controls. The activities of JNKs to phosphorylate GST-c-Jun were increased by 2-fold in ICM failing heart (P < .05), but not in DCM failing heart. These results selectively demonstrated that JNKs were upregulated and persistently activated in the ischemic 5tiling human heart. Given the fact that experimental myocardial ischemia followed by reperfusion induces activation of JNKs, the upregulation and persistent activation of JNKs in the ischemic failing human heart may be a result as well as an indicator of repeated ischemia with reperfusion.