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A Loss of Function Gq Promoter Polymorphism Is Associated with Accelerated Progression of Heart Failure
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Journal of Cardiac Failure Vol. 11 No. 6 Suppl. 2005
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022
Superior Natriuretic and Diuretic Actions of a Novel Long-Acting ANP Designer Peptide Compared to Native ANP in Normal Dogs Paul M. McKie1,2,3, Alessandro Cataliotti1, Fernando L. Martin1, Gail J. Harty1, Lynn K. Harstad1, John C. Burnett; 1Cardiorenal Research Laboratory, Mayo Clinic, Rochester, MN; 2Researh Fellow, Stanley J Sarnoff Endowment for Cardiovascular Research, Great Falls, VA; 3Dartmouth Medical School, Hanover, NH
MMP 13 Promoter Variant Is Associated with Increased Mortality and Heart Failure Progression Daniel L. Dries1, Philip Lavori1, Ken B. Margulies1, Surai Thaneemit-Chen2, Thomas P. Cappola2; 1Cardiology Division, Hospital of the University of Pennsylvania, Philadelphia, PA; 2Veteran’s Affairs DNA Bank Coordinating Center, Stanford, Menlo Park, CA
The cardiac natriuretic peptide ANP is a 28 amino acid (AA) peptide which consists of a 17 AA ring formed by a disulfide bond together with a 6 AA N-terminus and a 5 AA C-terminus. Studies suggest that the C-terminus is a key requirement for the natriuretic actions of ANP and that it also provides resistance to peptide degradation by neutral endopeptidase. Here we report a novel Mayo developed 40 AA peptide, termed Long-Acting ANP (LA-ANP), whose structure consists of native ANP 1–28 with an additional 12 AA C-terminus which we hypothesized would enhance the natriuretic and diuretic actions of ANP. Methods: Studies were performed in anesthetized normal dogs comparing equimolar concentrations (2 pmol/kg/min) of intravenous infusion of LA-ANP to ANP where ANP was administered at a threshold concentration which activated cGMP. Renal function was determined at baseline (BL), during 45 minutes of infusion, and 120 minutes post-infusion. Results: Overall, LAANP resulted in a greater diuretic and natriuretic action as compared to ANP (p ⬍ 0.01 for both). Specifically, natriuresis was not increased with ANP during infusion or at 120 minutes post-infusion. In contrast, LA-ANP significantly increased urinary sodium excretion from 47.0 ⫾ 18.9 µEq/min at BL to 88.4 ⫾ 17.5 µEq/min during infusion (p ⬍ 0.01), and to 119.2 ⫾ 34.2 µEq/min at 120 minutes post-infusion (p ⬍ 0.01). Similarly, there was no significant change in diuresis with native ANP but diuresis increased with LA-ANP from 0.34 ⫾ 0.08 ml/min at BL to 1.08 ⫾ 0.23 ml/min with infusion (p ⬍ 0.01). The natriuretic and diuretic actions of LA-ANP were localized to the terminal nephron where distal fractional sodium reabsorption decreased from 97.2 ⫾ 0.9% at BL to 96.0 ⫾ 0.6% during infusion and to 95.3 ⫾ 0.9% at 120 minutes post-infusion. Overall, this was significantly decreased when compared to native ANP (p ⬍ 0.01). No change in glomerular filtration rate, renal blood flow, or mean arterial pressure was observed during LA-ANP or ANP infusion. Conclusions: These studies report a novel 40 AA LA-ANP with an elongated C-terminus. This designer peptide possesses superior and sustained natriuretic and diuretic actions compared to native ANP and thus may serve as a potential novel saluretic and diuretic agent in sodium retaining syndromes.
Background: MMP 13 is the major collagenase activated in heart failure. MMP’s regulate extracellular matrix dynamics, including collagen turnover. MMP activity at the tissue level is primarily regulated at the level of transcription. Methods: The promoter region of the human MMP13 gene was sequenced in 30 African-Americans with NIDCM. The association of MMP13 promoter SNPs with prognosis in patients with heart failure was determined in participants (N ⫽ 1008) in the Bucindolol Evaluation and Survival Trial DNA-Substudy. Results: A novel SNP (A/T) was identifed located at position -449 in the MMP13 promoter, disrupting a consensus binding site for GATA transcription factors. Unadjusted survival analysis suggested a trend (P ⫽ 0.13) for a survival difference between the three MMP13-449 promoter genotype groups (P ⫽ 0.13); explained by a significantly higher mortality in the group homozygous for the minor MMP13-449 promoter allele (TT). The MMP13-449 (TT) group (138/969; 14%) compared to the combined group (AA or AT) (831/969; 86%) had similar baseline characteristics, except for a slightly increased prevalence of ischemic cardiomyopathy (67 vs. 57%; P ⫽ 0.04) and a lower proportion of selfreported African-Americans (13 vs. 23%; P ⫽ 0.01). Race was not independently associated with mortality in BEST. In unadjusted analysis, the MMP13-449 (TT) group compared to the combined group demonstrated an increased risk for all-cause mortality (ACM) (1.52; 95% CI 1.02–2.27; P ⫽ 0.04) and an increased risk for the composite end-point od death or CHF hospitalization (RR 1.41; 95% CI 1.07– 1.89; P ⫽ 0.016). Despite adjusting for difference in etiology (ischemic vs. nonischemic) and randomization assignment (bucindolol vs. placebo), the MMP13/449/ TT group demonstrated a strong trend for an increased risk for all-cause mortality (1.42; 95% 0.95- ⫽ 2.12; P ⫽ 0.09) and a significantly increased risk for the composite end-point (RR 1.37; 95% CI 1.03–1.81; P ⫽ 0.03).Conclusions. HF patients homozygous for the minor MMP13-449 (A/T) promoter allele demonstrate a trend for an increased risk for ACM mortality and a statistically significant increased risk the composite end-point of death or heart failure hospitalization. This may be explained by the MMP13-449 (A/T) SNP reducing MMP13 gene transcription by disrupting a GATA transcription factor binding site, resulting in collagen accumulation and enhanced cardiac fibrosis.
021
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Molecular Classification of Idiopathic Dilated Cardiomyopathy Thomas P. Cappola1, Mahlet G. Tadesse2, Mary E. Putt2, Joan Gilmore1, Daniel L. Dries1, Kenneth B. Margulies1; 1Cardiovascular Medicine, University of Pennsylvania, Philadelphia, PA; 2Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, PA
A Loss of Function Gq Promoter Polymorphism Is Associated with Accelerated Progression of Heart Failure Harvey S. Hahn1, Reagan J. Kelly2, Ulrich H. Frey3, Winfried Siffert3, Stephen B. Liggett1, Gerald W. Dorn II1; 1Division of Cardiology, University of Cincinnati, Cincinnati, OH; 2University of Michigan, Ann Arbor, MI; 3Institute of Pharmacology, Essen, Germany
Background: Idiopathic dilated cardiomyopathy (IDCM) has a clinically homogenous presentation despite evidence suggesting diverse etiologies (infection, inherited or spontaneous mutations, toxic exposures). We investigated whether sub-classes of idiopathic dilated cardiomyopathy, and the genes that characterize the proposed subclasses, could be simultaneously discerned through a Bayesian analysis of the myocardial transcriptome. We used a novel Markov chain Monte Carlo (MCMC) technique to simultaneously uncover subgroups and identify their discriminating genes. Methods and Results: Myocardial RNA samples were isolated from 78 patients with IDCM at the time of heart transplantation and were hybridized with individual Affymetrix HU133A arrays. Data were normalized and filtered using RMA (www.bioconductor. org) resulting in 13,639 transcripts present above background. We analyzed these transcripts using the MCMC method for class discovery and gene selection. MCMC is an iterative procedure that explores a wide range of possible models for class membership as well as the expression levels among genes that best discriminate the classes. Thus neither the number of classes nor the number of discriminating genes is determined in advance of the analysis. We ran six separate MCMC analyses of 50,000 iterations each with different initial sample allocations and discriminating genes. There was good concordance across all analyses, with convergence to models with between two and four distinct sub-classes of IDCM. Strongest support was found for the two sub-class model. Allocation of patients to the two sub-classes and the genes that best distinguish these two groups were determined based on their respective posterior probabilities. For the two sub-classes model, ten patients were allocated to a distinct sub-class of IDCM and 43 discriminating genes were selected on the basis of posterior probabilities in excess of 0.5. Conclusions: Our analysis provides strong evidence for two molecular sub-classes of IDCM which can be distinguished by a relatively small set of 43 myocardial transcripts. These findings suggest diversity in the pathologic features of IDCM beyond what can be discerned by standard clinical data. Further studies are necessary to determine if molecular subtypes of IDCM have different prognoses and respond differently to therapy.
Background: Gq is a critical regular of cardiac hypertrophy, which is an independent risk factor for death. Transgenic mouse studies have demonstrated that Gq is necessary for pressure-induced hypertrophy, sufficient to cause load-independent hypertrophy, and can mediate the transition of compensated hypertrophy to congestive heart failure (CHF). The effects of modulated Gq signaling in the human condition are unknown. We Hypothesized that promoter polymorphisms of the Gq gene that affect Gq transcription could act as genetic determinants of heart failure progression. Methods/Results: A dinucleotide polymorphism of the human Gq promoter (GC to TT at -695/-694) was identified. In vitro characterization revealed a loss of agonist-stimulated Gq transcriptional activity due to ablation of a Sp-1 binding site. Patients from the Cincinnati Heart Failure Study (CHF n ⫽ 1057; non-affected n ⫽ 278) were genotyped for this Gq variant. Allele frequencies between non-affected and CHF Caucasians were 0.44 and 0.48, and African-Americans (AA) were 0.32 and 0.35 respectively. Univariate and multivariate analyses demonstrated no association of the Gq polymorphism with death and/or transplant in Caucasians. However, in AA CHF, median survival was reduced from 72.8 months in GC homozygotes, to 62.8 months in TT heterozygotes, and 55.7 months in TT homozygotes (p ⫽ 0.0058). Using a model that included all univariate predictors of death (age, sex, b-blockers, ejection fraction, and the Gq polymorphism) the relative risk (RR) of death for TT homozygotes was 3.89 (p ⫽ 0.009) and for TT heterozygotes it was 2.39 (p ⫽ 0.046), revealing an allele dose-response effect. The RR of death due to lack of b-blockers therapy was 3.03 (p ⫽ 0.012), indicating that homozygousity for the Gq polymorphism was the single most powerful predictor of death in our AA CHF population. Summary: Polymorphic loss of normal transcriptional activation for the human Gq gene constitutes a novel genetic risk factor for adverse outcomes in the AA CHF population and reveals a critical role for Gq signaling in human cardiac homeostasis.
Journal of Cardiac Failure Vol. 11 No. 6 Suppl. 2005
020
022
Superior Natriuretic and Diuretic Actions of a Novel Long-Acting ANP Designer Peptide Compared to Native ANP in Normal Dogs Paul M. McKie1,2,3, Alessandro Cataliotti1, Fernando L. Martin1, Gail J. Harty1, Lynn K. Harstad1, John C. Burnett; 1Cardiorenal Research Laboratory, Mayo Clinic, Rochester, MN; 2Researh Fellow, Stanley J Sarnoff Endowment for Cardiovascular Research, Great Falls, VA; 3Dartmouth Medical School, Hanover, NH
MMP 13 Promoter Variant Is Associated with Increased Mortality and Heart Failure Progression Daniel L. Dries1, Philip Lavori1, Ken B. Margulies1, Surai Thaneemit-Chen2, Thomas P. Cappola2; 1Cardiology Division, Hospital of the University of Pennsylvania, Philadelphia, PA; 2Veteran’s Affairs DNA Bank Coordinating Center, Stanford, Menlo Park, CA
The cardiac natriuretic peptide ANP is a 28 amino acid (AA) peptide which consists of a 17 AA ring formed by a disulfide bond together with a 6 AA N-terminus and a 5 AA C-terminus. Studies suggest that the C-terminus is a key requirement for the natriuretic actions of ANP and that it also provides resistance to peptide degradation by neutral endopeptidase. Here we report a novel Mayo developed 40 AA peptide, termed Long-Acting ANP (LA-ANP), whose structure consists of native ANP 1–28 with an additional 12 AA C-terminus which we hypothesized would enhance the natriuretic and diuretic actions of ANP. Methods: Studies were performed in anesthetized normal dogs comparing equimolar concentrations (2 pmol/kg/min) of intravenous infusion of LA-ANP to ANP where ANP was administered at a threshold concentration which activated cGMP. Renal function was determined at baseline (BL), during 45 minutes of infusion, and 120 minutes post-infusion. Results: Overall, LAANP resulted in a greater diuretic and natriuretic action as compared to ANP (p ⬍ 0.01 for both). Specifically, natriuresis was not increased with ANP during infusion or at 120 minutes post-infusion. In contrast, LA-ANP significantly increased urinary sodium excretion from 47.0 ⫾ 18.9 µEq/min at BL to 88.4 ⫾ 17.5 µEq/min during infusion (p ⬍ 0.01), and to 119.2 ⫾ 34.2 µEq/min at 120 minutes post-infusion (p ⬍ 0.01). Similarly, there was no significant change in diuresis with native ANP but diuresis increased with LA-ANP from 0.34 ⫾ 0.08 ml/min at BL to 1.08 ⫾ 0.23 ml/min with infusion (p ⬍ 0.01). The natriuretic and diuretic actions of LA-ANP were localized to the terminal nephron where distal fractional sodium reabsorption decreased from 97.2 ⫾ 0.9% at BL to 96.0 ⫾ 0.6% during infusion and to 95.3 ⫾ 0.9% at 120 minutes post-infusion. Overall, this was significantly decreased when compared to native ANP (p ⬍ 0.01). No change in glomerular filtration rate, renal blood flow, or mean arterial pressure was observed during LA-ANP or ANP infusion. Conclusions: These studies report a novel 40 AA LA-ANP with an elongated C-terminus. This designer peptide possesses superior and sustained natriuretic and diuretic actions compared to native ANP and thus may serve as a potential novel saluretic and diuretic agent in sodium retaining syndromes.
Background: MMP 13 is the major collagenase activated in heart failure. MMP’s regulate extracellular matrix dynamics, including collagen turnover. MMP activity at the tissue level is primarily regulated at the level of transcription. Methods: The promoter region of the human MMP13 gene was sequenced in 30 African-Americans with NIDCM. The association of MMP13 promoter SNPs with prognosis in patients with heart failure was determined in participants (N ⫽ 1008) in the Bucindolol Evaluation and Survival Trial DNA-Substudy. Results: A novel SNP (A/T) was identifed located at position -449 in the MMP13 promoter, disrupting a consensus binding site for GATA transcription factors. Unadjusted survival analysis suggested a trend (P ⫽ 0.13) for a survival difference between the three MMP13-449 promoter genotype groups (P ⫽ 0.13); explained by a significantly higher mortality in the group homozygous for the minor MMP13-449 promoter allele (TT). The MMP13-449 (TT) group (138/969; 14%) compared to the combined group (AA or AT) (831/969; 86%) had similar baseline characteristics, except for a slightly increased prevalence of ischemic cardiomyopathy (67 vs. 57%; P ⫽ 0.04) and a lower proportion of selfreported African-Americans (13 vs. 23%; P ⫽ 0.01). Race was not independently associated with mortality in BEST. In unadjusted analysis, the MMP13-449 (TT) group compared to the combined group demonstrated an increased risk for all-cause mortality (ACM) (1.52; 95% CI 1.02–2.27; P ⫽ 0.04) and an increased risk for the composite end-point od death or CHF hospitalization (RR 1.41; 95% CI 1.07– 1.89; P ⫽ 0.016). Despite adjusting for difference in etiology (ischemic vs. nonischemic) and randomization assignment (bucindolol vs. placebo), the MMP13/449/ TT group demonstrated a strong trend for an increased risk for all-cause mortality (1.42; 95% 0.95- ⫽ 2.12; P ⫽ 0.09) and a significantly increased risk for the composite end-point (RR 1.37; 95% CI 1.03–1.81; P ⫽ 0.03).Conclusions. HF patients homozygous for the minor MMP13-449 (A/T) promoter allele demonstrate a trend for an increased risk for ACM mortality and a statistically significant increased risk the composite end-point of death or heart failure hospitalization. This may be explained by the MMP13-449 (A/T) SNP reducing MMP13 gene transcription by disrupting a GATA transcription factor binding site, resulting in collagen accumulation and enhanced cardiac fibrosis.
021
023
Molecular Classification of Idiopathic Dilated Cardiomyopathy Thomas P. Cappola1, Mahlet G. Tadesse2, Mary E. Putt2, Joan Gilmore1, Daniel L. Dries1, Kenneth B. Margulies1; 1Cardiovascular Medicine, University of Pennsylvania, Philadelphia, PA; 2Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, Philadelphia, PA
A Loss of Function Gq Promoter Polymorphism Is Associated with Accelerated Progression of Heart Failure Harvey S. Hahn1, Reagan J. Kelly2, Ulrich H. Frey3, Winfried Siffert3, Stephen B. Liggett1, Gerald W. Dorn II1; 1Division of Cardiology, University of Cincinnati, Cincinnati, OH; 2University of Michigan, Ann Arbor, MI; 3Institute of Pharmacology, Essen, Germany
Background: Idiopathic dilated cardiomyopathy (IDCM) has a clinically homogenous presentation despite evidence suggesting diverse etiologies (infection, inherited or spontaneous mutations, toxic exposures). We investigated whether sub-classes of idiopathic dilated cardiomyopathy, and the genes that characterize the proposed subclasses, could be simultaneously discerned through a Bayesian analysis of the myocardial transcriptome. We used a novel Markov chain Monte Carlo (MCMC) technique to simultaneously uncover subgroups and identify their discriminating genes. Methods and Results: Myocardial RNA samples were isolated from 78 patients with IDCM at the time of heart transplantation and were hybridized with individual Affymetrix HU133A arrays. Data were normalized and filtered using RMA (www.bioconductor. org) resulting in 13,639 transcripts present above background. We analyzed these transcripts using the MCMC method for class discovery and gene selection. MCMC is an iterative procedure that explores a wide range of possible models for class membership as well as the expression levels among genes that best discriminate the classes. Thus neither the number of classes nor the number of discriminating genes is determined in advance of the analysis. We ran six separate MCMC analyses of 50,000 iterations each with different initial sample allocations and discriminating genes. There was good concordance across all analyses, with convergence to models with between two and four distinct sub-classes of IDCM. Strongest support was found for the two sub-class model. Allocation of patients to the two sub-classes and the genes that best distinguish these two groups were determined based on their respective posterior probabilities. For the two sub-classes model, ten patients were allocated to a distinct sub-class of IDCM and 43 discriminating genes were selected on the basis of posterior probabilities in excess of 0.5. Conclusions: Our analysis provides strong evidence for two molecular sub-classes of IDCM which can be distinguished by a relatively small set of 43 myocardial transcripts. These findings suggest diversity in the pathologic features of IDCM beyond what can be discerned by standard clinical data. Further studies are necessary to determine if molecular subtypes of IDCM have different prognoses and respond differently to therapy.
Background: Gq is a critical regular of cardiac hypertrophy, which is an independent risk factor for death. Transgenic mouse studies have demonstrated that Gq is necessary for pressure-induced hypertrophy, sufficient to cause load-independent hypertrophy, and can mediate the transition of compensated hypertrophy to congestive heart failure (CHF). The effects of modulated Gq signaling in the human condition are unknown. We Hypothesized that promoter polymorphisms of the Gq gene that affect Gq transcription could act as genetic determinants of heart failure progression. Methods/Results: A dinucleotide polymorphism of the human Gq promoter (GC to TT at -695/-694) was identified. In vitro characterization revealed a loss of agonist-stimulated Gq transcriptional activity due to ablation of a Sp-1 binding site. Patients from the Cincinnati Heart Failure Study (CHF n ⫽ 1057; non-affected n ⫽ 278) were genotyped for this Gq variant. Allele frequencies between non-affected and CHF Caucasians were 0.44 and 0.48, and African-Americans (AA) were 0.32 and 0.35 respectively. Univariate and multivariate analyses demonstrated no association of the Gq polymorphism with death and/or transplant in Caucasians. However, in AA CHF, median survival was reduced from 72.8 months in GC homozygotes, to 62.8 months in TT heterozygotes, and 55.7 months in TT homozygotes (p ⫽ 0.0058). Using a model that included all univariate predictors of death (age, sex, b-blockers, ejection fraction, and the Gq polymorphism) the relative risk (RR) of death for TT homozygotes was 3.89 (p ⫽ 0.009) and for TT heterozygotes it was 2.39 (p ⫽ 0.046), revealing an allele dose-response effect. The RR of death due to lack of b-blockers therapy was 3.03 (p ⫽ 0.012), indicating that homozygousity for the Gq polymorphism was the single most powerful predictor of death in our AA CHF population. Summary: Polymorphic loss of normal transcriptional activation for the human Gq gene constitutes a novel genetic risk factor for adverse outcomes in the AA CHF population and reveals a critical role for Gq signaling in human cardiac homeostasis.