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Gene Variant of the Bradykinin B2 Receptor Influences Pulmonary Arterial Pressures in Heart Failure Patients
S44 Journal of Cardiac Failure Vol. 12 No. 6 Suppl. 2006 143 Serum Response Factor Is Regionally Expressed in Ischemic Cardiomyopathy Rita Karianna C. Milewski1, Hirotsugu Hamamoto1, Shinya Kanemoto1, Camille Immanuel1, Theodore Plappert1, Michael S. Parmacek1, Joseph H. Gorman1, Robert C. Gorman1; 1Harrison Department of Surgical Research, University of Pennsylvania, Philadelphia, PA Background: Regionally heterogeneous changes in myocyte and interstitial structure and function occur during infarction (MI) induced left ventricular (LV) remodeling. Delineation of the molecular mechanisms responsible for this phenomenon is at present limited. Serum response factor, SRF, a platform transcription factor, has a central role in contractile protein regulation in the adult heart. In mouse models and mixed cardiomyopathic human tissue samples, four major isoforms of SRF have been identified. Studies suggest the 55kDa and 32kDa isoforms repress myocardial gene expression. We postulate that regional differential expression of SRF isoforms may contribute to the myopathic process that occurs as the LV remodels after an MI. Methods: Using an ovine model of anteroapical infarction, myocardium was harvested from three regions of the LV; remote, borderzone, and infarct (n 5 4) at eight weeks after MI. Normal sheep were used as controls (n 5 4). Anti-SRF antibody (Santa Cruz) was used to perform protein analysis of SRF isoforms in each myocardial sample. Equal amounts of protein were used from each myocardial sample. Remodeling was assessed with echocardiography. Results: SRF isoform protein levels were examined in each sample. Full length SRF (SRF-FL) (65kDa) was intact in normal samples. SRF-FL was decreased in the borderzone region at eight weeks accompanied by an increase in the 55kDa isoform. SRF-FL was replaced in the infarct region by the 55kDa isoform and 32kDa isoform. All animals developed severe LV dilatation by the eight week follow-up time point.[figure1] Conclusion: SRF isoforms are regionally expressed in the remodeled LV. There is a progression from full length functional protein in normal myocardium to the 55kDa and 32kDa isoforms in the normally perfused borderzone and infarct myocardium. Regional and temporal differences in the expression of SRF isoforms may contribute to the inherent myopathic process associated with infarction induced remodeling.
were male, 31% had ischemic HF. 46 subjects (35%) were homozygous for the þ9 allele, 58 (44%) were heterozygous (þ9/-9) and 27 (21%) were homozygous for the -9 allele of the B2R. RV systolic pressure averaged 42 6 13, 38 6 12, and 35 6 11 mmHg for the þ9/þ9, þ9/-9 and -9/-9 subjects respectively, p 5 0.03 for trend. Although ACE values were skewed towards 0 due to chronic ACE suppression, there was a tendency for a gene effect on plasma ACE levels with the highest values observed in the þ9/þ9 subjects, median 5 5U/L (5th% 5 1, 95th% 5 28U/L), and least in the -9/-9 subjects, median 5 2U/L (5th% 5 0, 95th% 5 17U/L), p 5 0.06, however, there were no significant differences in plasma bradykinin or angiotensin II values or other neurohormones according to genotype (pO0.10). Similarly, LVEF and NYHA class did not differ significantly across the genotype groups. Conclusion: These data suggest that the þ9/-9 polymorphism of the bradykinin B2 receptor may influence pulmonary vascular tone in patients with stable HF.
145 Gene Expression Changes in Isolated Human Ventricular Preparations Treated with the b-Adrenergic Receptor Agonist Isoproterenol Carmen C. Sucharov1, Bradley Nelson1, Jennifer Morrison1, Wayne Minobe1, Michael R. Bristow1; 1Cardiology, Universitry of Colorado Health Sciences Center, Denver, CO b-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathy (DCM), exerting both compensatory effects on cardiac function and promoting the development and progression of the DCM phenotype. Activation of b-adrenergic receptors (b1-AR and b2-AR) during periods of cardiac stress initially results in increases in heart rate and contractility, effectively improving cardiac output, but then ultimately harms the failing heart by altering gene expression patterns and cardiac morphology. Of the changes that are observed in failing hearts, increases in b myosin heavy chain (bMyHC), skeletal a-actin, and atrial natriuretic peptide (ANP), with coordinate decreases in a myosin heavy chain (aMyHC) and sarcoplasmatic reticulum ATPase 2a (SRCA2a), are perhaps the most widely recognized. Gene expression studies in response to b-AR stimulation have been done mostly in animal models, with the exception of one study performed by our group (Lowes et al), in which treatment of heart failure patients with b-blocker therapy results in a partial reversal of the fetal gene program. Here we show that by treating isolated human trabeculae with the b-AR agonist isoproterenol for 48 hours, changes in the fetal gene program are observed, with up-regulation of ANF, BNP and skeletal a-actin. Briefly, individual RV trabeculae of uniform size (1 to 2 by 6 to 8 mm) were isolated from the free wall of the right ventricle and placed in muscle bath chambers containing Tyrode’s buffer equilibrated with 95% O2/5% CO2 and kept at 37 C. Trabeculae were suspended between plastic mounting clips and treated with 10-7M isoproterenol for 48 hours, and gene expression was measured by RT-PCR. These results suggest that human trabeculae are a valid model system to analyze changes in gene expression in response to hypertrophic agonists in human subjects.
146 Ventricular Remodeling in Infarcted Mouse Hearts Can Be Controlled by Molecular Induction of Intracardiac Graft Cell Proliferation Kelly R. Stevens1, Marsha W. Rolle2, Elina Minami3, Shichi Ueno3, Jitka I. Virag4, Marilyn B. Nourse1, Lil Pabon5, Hans Reinecke5, Charles E. Murry5; 1 Bioengineering, University of Washington, Seattle, WA; 2Benaroya Research Institute, Seattle, WA; 3Medicine, University of Washington, Seattle, WA; 4 Physiology, East Carolina University, Greenville, NC; 5Pathology, University of Washington, Seattle, WA
144 Gene Variant of the Bradykinin B2 Receptor Influences Pulmonary Arterial Pressures in Heart Failure Patients Thomas P. Olson1, Eric M. Snyder1, Robert P. Frantz1, Minelle L. Hulsebus1, Kathy A. O’Malley1, Kent R. Bailey1, Christina M. Wood1, Lyle J. Olson1, Steve T. Turner1, Bruce D. Johnson1; 1Cardiovascular Diseases, Mayo Clinic, Rochester, MN Background: Pulmonary arterial pressures (PAP) vary considerably in patients with heart failure (HF) despite similar degrees of left ventricular (LV) dysfunction. Alterations in the renin-angiotensin system may play a role in modulating pulmonary vascular tone. A common gene variant in the kinin B2 receptor (B2R) exists: the -9 as opposed to the þ9 allele is associated with higher receptor mRNA expression. Most of the physiological function of bradykinin is likely mediated through this receptor, including vasodilatation. We sought to determine if genetic variation in this receptor influences PAP in a well defined HF population. Methods: Patients with O1 yr. history of systolic HF, ischemic and dilated etiologies, without COPD and not currently smoking, BMI !40, without atrial fibrillation were recruited. A total of 131 subjects completed the study, which included a blood draw for genotyping and neurohormones (ACE, A-II, Bradykinin, ANP, BNP, catecholamines), an echocardiogram for assessment of systolic and diastolic function as well as estimates of RV systolic pressure (tricuspid regurgitant velocity). Results: Average LVEF for the group was 29 6 12%, NYHA class 2 6 1, age 56 6 12 yr., BMI 28 6 5 kg/m2, 65% of patients
Enhanced proliferation of cell grafts in the heart and consequent augmentation of graft size could dramatically improve cell therapies for cardiac repair. In order to selectively stimulate the proliferation of graft cells, we created a chimeric receptor composed of a modified FK506-binding protein (F36V) fused with the cytoplasmic domain of the fibroblast growth factor receptor-1 (F36V-fgfr1). We retrovirally transduced mouse C2C12 and MM14 skeletal myoblasts with this construct and treated them with AP20187, a dimeric F36V ligand (‘‘dimerizer’’), in vitro and in vivo to induce receptor dimerization. Treatment of myoblasts expressing F36Vfgfr-1 with dimerizer activated the MAP kinase pathway and induced proliferation comparable to treatment with bFGF. Wild type myoblasts did not proliferate in response to dimerizer in vitro. Subcutaneous grafts of myoblasts expressing F36Vfgfr-1 showed a dosedependent increase in DNA synthesis in response to dimerizer compared to mice receiving vehicle only. When F36Vfgfr-1 expressing myoblasts were injected into infarcted hearts of nude mice, treatment with dimerizer resulted in significantly larger grafts (p ! 0.01). Specifically, intracardiac C2C12 F36Vfgfr-1 grafts identified by embryonic myosin staining occupied 42.9 6 4.3%, 40.4 6 6.8%, or 20 6 3% of the left ventricle in mice receiving 24 mg/kg/day, 9.6 mg/kg/day, or 0 mg/kg/day dimerizer, respectively. Echocardiography demonstrated that larger graft size was associated with a dose-dependent reduction in ventricular dilation following myocardial infarction. Thus, selective proliferation of modified graft cells was induced with a systemically administered small molecule and controlled ventricular remodeling. Human-embryonic stem cells (huESC) were recently lentivirally transduced with a chimeric receptor composed of F36V fused with the cytoplasmic domain of the insulin-derived growth factor receptor-1 (F36Vigfr-1). HuESC-derived cardiomyocytes expressing F36Vigfr-1 will be treated with dimerizer to control huESC-derived cardiomyocyte proliferation. This approach allows fine-tuning of intracardiac graft size to attain a desired physiological outcome.
were male, 31% had ischemic HF. 46 subjects (35%) were homozygous for the þ9 allele, 58 (44%) were heterozygous (þ9/-9) and 27 (21%) were homozygous for the -9 allele of the B2R. RV systolic pressure averaged 42 6 13, 38 6 12, and 35 6 11 mmHg for the þ9/þ9, þ9/-9 and -9/-9 subjects respectively, p 5 0.03 for trend. Although ACE values were skewed towards 0 due to chronic ACE suppression, there was a tendency for a gene effect on plasma ACE levels with the highest values observed in the þ9/þ9 subjects, median 5 5U/L (5th% 5 1, 95th% 5 28U/L), and least in the -9/-9 subjects, median 5 2U/L (5th% 5 0, 95th% 5 17U/L), p 5 0.06, however, there were no significant differences in plasma bradykinin or angiotensin II values or other neurohormones according to genotype (pO0.10). Similarly, LVEF and NYHA class did not differ significantly across the genotype groups. Conclusion: These data suggest that the þ9/-9 polymorphism of the bradykinin B2 receptor may influence pulmonary vascular tone in patients with stable HF.
145 Gene Expression Changes in Isolated Human Ventricular Preparations Treated with the b-Adrenergic Receptor Agonist Isoproterenol Carmen C. Sucharov1, Bradley Nelson1, Jennifer Morrison1, Wayne Minobe1, Michael R. Bristow1; 1Cardiology, Universitry of Colorado Health Sciences Center, Denver, CO b-adrenergic signaling plays an important role in the natural history of dilated cardiomyopathy (DCM), exerting both compensatory effects on cardiac function and promoting the development and progression of the DCM phenotype. Activation of b-adrenergic receptors (b1-AR and b2-AR) during periods of cardiac stress initially results in increases in heart rate and contractility, effectively improving cardiac output, but then ultimately harms the failing heart by altering gene expression patterns and cardiac morphology. Of the changes that are observed in failing hearts, increases in b myosin heavy chain (bMyHC), skeletal a-actin, and atrial natriuretic peptide (ANP), with coordinate decreases in a myosin heavy chain (aMyHC) and sarcoplasmatic reticulum ATPase 2a (SRCA2a), are perhaps the most widely recognized. Gene expression studies in response to b-AR stimulation have been done mostly in animal models, with the exception of one study performed by our group (Lowes et al), in which treatment of heart failure patients with b-blocker therapy results in a partial reversal of the fetal gene program. Here we show that by treating isolated human trabeculae with the b-AR agonist isoproterenol for 48 hours, changes in the fetal gene program are observed, with up-regulation of ANF, BNP and skeletal a-actin. Briefly, individual RV trabeculae of uniform size (1 to 2 by 6 to 8 mm) were isolated from the free wall of the right ventricle and placed in muscle bath chambers containing Tyrode’s buffer equilibrated with 95% O2/5% CO2 and kept at 37 C. Trabeculae were suspended between plastic mounting clips and treated with 10-7M isoproterenol for 48 hours, and gene expression was measured by RT-PCR. These results suggest that human trabeculae are a valid model system to analyze changes in gene expression in response to hypertrophic agonists in human subjects.
146 Ventricular Remodeling in Infarcted Mouse Hearts Can Be Controlled by Molecular Induction of Intracardiac Graft Cell Proliferation Kelly R. Stevens1, Marsha W. Rolle2, Elina Minami3, Shichi Ueno3, Jitka I. Virag4, Marilyn B. Nourse1, Lil Pabon5, Hans Reinecke5, Charles E. Murry5; 1 Bioengineering, University of Washington, Seattle, WA; 2Benaroya Research Institute, Seattle, WA; 3Medicine, University of Washington, Seattle, WA; 4 Physiology, East Carolina University, Greenville, NC; 5Pathology, University of Washington, Seattle, WA
144 Gene Variant of the Bradykinin B2 Receptor Influences Pulmonary Arterial Pressures in Heart Failure Patients Thomas P. Olson1, Eric M. Snyder1, Robert P. Frantz1, Minelle L. Hulsebus1, Kathy A. O’Malley1, Kent R. Bailey1, Christina M. Wood1, Lyle J. Olson1, Steve T. Turner1, Bruce D. Johnson1; 1Cardiovascular Diseases, Mayo Clinic, Rochester, MN Background: Pulmonary arterial pressures (PAP) vary considerably in patients with heart failure (HF) despite similar degrees of left ventricular (LV) dysfunction. Alterations in the renin-angiotensin system may play a role in modulating pulmonary vascular tone. A common gene variant in the kinin B2 receptor (B2R) exists: the -9 as opposed to the þ9 allele is associated with higher receptor mRNA expression. Most of the physiological function of bradykinin is likely mediated through this receptor, including vasodilatation. We sought to determine if genetic variation in this receptor influences PAP in a well defined HF population. Methods: Patients with O1 yr. history of systolic HF, ischemic and dilated etiologies, without COPD and not currently smoking, BMI !40, without atrial fibrillation were recruited. A total of 131 subjects completed the study, which included a blood draw for genotyping and neurohormones (ACE, A-II, Bradykinin, ANP, BNP, catecholamines), an echocardiogram for assessment of systolic and diastolic function as well as estimates of RV systolic pressure (tricuspid regurgitant velocity). Results: Average LVEF for the group was 29 6 12%, NYHA class 2 6 1, age 56 6 12 yr., BMI 28 6 5 kg/m2, 65% of patients
Enhanced proliferation of cell grafts in the heart and consequent augmentation of graft size could dramatically improve cell therapies for cardiac repair. In order to selectively stimulate the proliferation of graft cells, we created a chimeric receptor composed of a modified FK506-binding protein (F36V) fused with the cytoplasmic domain of the fibroblast growth factor receptor-1 (F36V-fgfr1). We retrovirally transduced mouse C2C12 and MM14 skeletal myoblasts with this construct and treated them with AP20187, a dimeric F36V ligand (‘‘dimerizer’’), in vitro and in vivo to induce receptor dimerization. Treatment of myoblasts expressing F36Vfgfr-1 with dimerizer activated the MAP kinase pathway and induced proliferation comparable to treatment with bFGF. Wild type myoblasts did not proliferate in response to dimerizer in vitro. Subcutaneous grafts of myoblasts expressing F36Vfgfr-1 showed a dosedependent increase in DNA synthesis in response to dimerizer compared to mice receiving vehicle only. When F36Vfgfr-1 expressing myoblasts were injected into infarcted hearts of nude mice, treatment with dimerizer resulted in significantly larger grafts (p ! 0.01). Specifically, intracardiac C2C12 F36Vfgfr-1 grafts identified by embryonic myosin staining occupied 42.9 6 4.3%, 40.4 6 6.8%, or 20 6 3% of the left ventricle in mice receiving 24 mg/kg/day, 9.6 mg/kg/day, or 0 mg/kg/day dimerizer, respectively. Echocardiography demonstrated that larger graft size was associated with a dose-dependent reduction in ventricular dilation following myocardial infarction. Thus, selective proliferation of modified graft cells was induced with a systemically administered small molecule and controlled ventricular remodeling. Human-embryonic stem cells (huESC) were recently lentivirally transduced with a chimeric receptor composed of F36V fused with the cytoplasmic domain of the insulin-derived growth factor receptor-1 (F36Vigfr-1). HuESC-derived cardiomyocytes expressing F36Vigfr-1 will be treated with dimerizer to control huESC-derived cardiomyocyte proliferation. This approach allows fine-tuning of intracardiac graft size to attain a desired physiological outcome.