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Cofilin-1: A Possible New Biomarker for Human Heart Failure
The 11th Annual Scientific Meeting
HFSA
S101
Neurohormones/Cytokines 093 Cofilin-1: A Possible New Biomarker for Human Heart Failure Brenda K. Huntley1, Denise M. Heublein1, Sharon M. Sandberg1, John C. Burnett, Jr.1; 1Cardiorenal Research, Mayo Clinic and Mayo Clinic College of Medicine, Rochester, MN Background: Heart Failure (HF) is characterized by activation of the natriuretic peptides ANP and BNP. In particular, BNP has emerged as a useful biomarker in human HF. While evaluating molecular forms of BNP in human plasma, we identified a new protein in plasma of patients with HF and elevated plasma BNP. Here we report the methodology and identification of this novel protein. Methods: Plasma from normal (n 5 5) and HF patients (n 5 10) was first subjected to albumin depletion using the SwellGel Blue Albumin Removal Kit (Pierce, Rockford, IL). For protein detection, gels were stained with Coomassie blue stain (Bio-Rad, Hercules, CA). Protein sequencing was performed by the Mayo Proteomics Core Facility. Western blotting was performed on PVDF membranes. Results: Albumin depleted human plasma on Coomassie blue stained gels revealed an approximately 20kDa protein band in HF plasma which was not evident in normal plasma samples. This band was sequenced and by database analysis determined to be Cofilin-1. The identity of the band was verified by western blot of plasma samples for Cofilin-1. To determine whether the Cofilin-1 identified was the active and/or inactive form of Cofilin-1, the western blots were stripped and re-probed using an antibody specific for the phosphorylated form of Cofilin-1. No bands were detected suggesting that the Cofilin-1 expressed in HF plasma is active dephosphorylated Cofilin-1. Here we identify exclusively in the plasma of HF patients, the actin binding protein Cofilin-1. Cofilin-1 is a ubiquitously expressed actin-associated protein which serves as an important convergent point in the cell signaling network through which a variety of extracellular stimuli regulate actin cytoskeletal dynamics and organization. It has been shown to play a role in cell cycle progression with over-expression leading to cell cycle withdraw at G1. In cardiomyocytes, Cofilin-1 expression and function has been linked to hypertrophic responses and in a recent proteomic analysis of cardiomyocytes subjected to proteasome inhibition, Cofilin-1 was one of nine proteins to be upregulated at both the protein and mRNA level in a manner similar to ANP. Conclusion: Our studies therefore identify for the first time that Cofilin-1 is present in human HF plasma while it is not found in normal plasma. We further demonstrate that Cofilin-1 in HF patients is in its active form. Additional studies are warranted to determine the utility of Cofilin-1 as a possible biomarker for HF and other cardiovascular syndromes.
094 Accelerated Decline in Circulating Progenitor Cells Due to Aldosterone Excess in Experimental Hypertension Barry A. Boilson1, Brian P. Shapiro1, Donna M. Meyer1, Adriana Harbuzariu1, Cheryl S. Mueske1, James E. Tarara1, Robert D. Simari1, Margaret M. Redfield1; 1 Division of Cardiovascular Diseases, Mayo Clinic College of Medicine, Rochester, MN Introduction: Bone marrow-derived progenitor cells may have a reparative and thus potentially therapeutic role in cardiovascular disease. Reduced reparative progenitor cell production or function may play a role in hypertension related vascular injury. Further, in vitro studies suggest that humoral activation (aldosterone) impairs production of progenitor cells, potentially limiting endogenous repair mechanisms and accelerating vascular damage during the transition to heart failure. Hypothesis: We hypothesized that circulating progenitor cells would be depleted in vivo in response to experimental hypertension and that these changes would be accelerated in the presence of concomitant aldosterone excess. Methods: Ten dogs underwent renal wrapping (RW) to induce hypertension. Blood pressure (BP) was measured weekly. After development of hypertension (5 weeks after RW), dogs were randomized to receive deoxycorticosterone acetate (1mg/kg/day for 3 weeks; HTN þ DOCA) or not (HTN). Flow cytometric analysis for CD34þ (progenitor) cells was performed on whole blood harvested prior to and every 2 weeks after RW. ISHAGE criteria were used to enumerate CD34þ cells. Results: See Figure. Dogs were similarly hypertensive at 5 weeks; BP increased week 5-8 only in HTN þ DOCA dogs. The CD34þ cell counts were similar prior to RW and decreased over time after RW in both groups. However, the decline in CD34þ cells was accelerated in the HTN þ DOCA dogs after DOCA treatment. Conclusion: This study demonstrates a progressive decrease in circulating progenitor cells over time after the onset of experimental hypertension. This may represent impaired bone marrow production or homing to sites of hypertension related vascular injury. However, the dramatic drop in CD34þ cells after DOCA administration is consistent with the previous in vitro studies suggesting that aldosterone impairs bone marrow derived progenitor cell formation. These data support the concept that humoral activation accelerates vascular injury by limiting endogenous reparative mechanisms.
095 Interferon Regulatory Factor-9 (IRF-9) Mediates Short Term Host Protection, but Promotes Long Term Immune Injury in Evolution of Myocarditis Leading to Dilated Cardiomyopathy Michael Konviser1,2, Youan Liu1, Manyin Chen1, Mei Sun1, Yu Shi1, Anthony Gramolini2, Anne Opavsky2, Eleanor Fish2, Peter Liu1,2,3; 1Toronto General Reasearch Institute; 2University of Toronto; 3Canadian Institute of Health Reasearch Introduction: Evolution of viral myocarditis to dilated cardiomyopathy (DCM) represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. However, the influence of innate immunity in general and IRF-9 in particular on acquired immunity and DCM is unknown. Hypothesis: IRF-9 signaling provides immediate host protection through interferon production, but stimulates acquired immunity leading to DCM in a coxsackievirus murine myocarditis model. Methods: Interferon-regulatory factor 9 (IRF-9) homozygous knockout mice were generated, and show impaired type I interferon production. Wild-type (WT, n 5 51) and IRF-9-/- (n 5 124) littermates were inoculated with 10^2 p.f.u. of coxsackievirus B3 as previously described. Survival, viral titers, cardiac hypertrophy, inflammation and fibrosis were evaluated on days 0, 4, 7, 10, 14, 28 and 42. Splenocyte subpopulations were cell sorted and quantitated by FACS. Results: IRF9-/- mice showed dramatically increased mortality compared to the wild-type littermates (0% WT vs 72% IRF-9-/- on day 14, P ! 0.0001). On day 42, there was less cardiac hypertrophy in IRF-9-/- mice compared to WT controls (p ! 0.05). There was no difference in inflammation and fibrosis. The mature T-lymphocyte population, defined as CD4 or CD8 single positive, was statistically identical in the two populations up until and including day 28 post-infection. However on day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9-/- spleens (p ! 0.05). Conclusions: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.
096 Identification of Genetic Variants of the Tumor Necrosis Factor Receptor 2 Gene (TNFRSF1B) in Patients with Heart Failure Akash Makkar1, Javier Banchs1, Mary A. Hauck2, Tariq S. Siddiqui1, Saeed A. Jortani3, Roland Valdes3, Steven C. Koenig2, Michael E. Mitchell4, Aoy TomitaMitchell4, Sumanth D. Prabhu1; 1Medicine, University of Louisville; 2Surgery, University of Louisville; 3Pathology, University of Louisville; 4Surgery, Medical College of Wisconsin Background: Tumor necrosis factor-alpha (TNF) plays an important pathophysiological role in heart failure (HF). As TNF signals exclusively via its two receptors (TNFR1 and TNFR2), TNFR polymorphisms may modulate the biological effects of TNF in HF and impact disease progression. The gene for TNFR2, TNFRSF1B, has been implicated through linkage studies in the pathogenesis of hypertension and coronary artery disease. However, whether TNFRSF1B is similarly linked to the pathogenesis of HF is unknown. Methods: To test the hypothesis that TNFRSF1B mutations contribute to HF, we conducted a pilot study in patients with systolic HF (LVEF ! 40%, NYHA class II-IV; excluded if recent [! 3 mo] infarction, significant valvular disease, or acute infection) to screen for mutations in the coding and flanking introns of TNFRSF1B and compared them to healthy controls without HF using heteroduplex analysis by capillary electrophoresis as well as direct sequencing. Results: We identified 3 previously described SNPs (K56K, M196R, E232K), 2 intronic mutations, and a novel synonymous mutation (A19A, patient with non-ischemic HF) in the coding region. The previously described SNPs occurred as haplotypes with the M196R SNP. The M196R SNP, previously reported to be associated with systemic lupus erythematosus (among other disorders), was found to occur at 51%
HFSA
S101
Neurohormones/Cytokines 093 Cofilin-1: A Possible New Biomarker for Human Heart Failure Brenda K. Huntley1, Denise M. Heublein1, Sharon M. Sandberg1, John C. Burnett, Jr.1; 1Cardiorenal Research, Mayo Clinic and Mayo Clinic College of Medicine, Rochester, MN Background: Heart Failure (HF) is characterized by activation of the natriuretic peptides ANP and BNP. In particular, BNP has emerged as a useful biomarker in human HF. While evaluating molecular forms of BNP in human plasma, we identified a new protein in plasma of patients with HF and elevated plasma BNP. Here we report the methodology and identification of this novel protein. Methods: Plasma from normal (n 5 5) and HF patients (n 5 10) was first subjected to albumin depletion using the SwellGel Blue Albumin Removal Kit (Pierce, Rockford, IL). For protein detection, gels were stained with Coomassie blue stain (Bio-Rad, Hercules, CA). Protein sequencing was performed by the Mayo Proteomics Core Facility. Western blotting was performed on PVDF membranes. Results: Albumin depleted human plasma on Coomassie blue stained gels revealed an approximately 20kDa protein band in HF plasma which was not evident in normal plasma samples. This band was sequenced and by database analysis determined to be Cofilin-1. The identity of the band was verified by western blot of plasma samples for Cofilin-1. To determine whether the Cofilin-1 identified was the active and/or inactive form of Cofilin-1, the western blots were stripped and re-probed using an antibody specific for the phosphorylated form of Cofilin-1. No bands were detected suggesting that the Cofilin-1 expressed in HF plasma is active dephosphorylated Cofilin-1. Here we identify exclusively in the plasma of HF patients, the actin binding protein Cofilin-1. Cofilin-1 is a ubiquitously expressed actin-associated protein which serves as an important convergent point in the cell signaling network through which a variety of extracellular stimuli regulate actin cytoskeletal dynamics and organization. It has been shown to play a role in cell cycle progression with over-expression leading to cell cycle withdraw at G1. In cardiomyocytes, Cofilin-1 expression and function has been linked to hypertrophic responses and in a recent proteomic analysis of cardiomyocytes subjected to proteasome inhibition, Cofilin-1 was one of nine proteins to be upregulated at both the protein and mRNA level in a manner similar to ANP. Conclusion: Our studies therefore identify for the first time that Cofilin-1 is present in human HF plasma while it is not found in normal plasma. We further demonstrate that Cofilin-1 in HF patients is in its active form. Additional studies are warranted to determine the utility of Cofilin-1 as a possible biomarker for HF and other cardiovascular syndromes.
094 Accelerated Decline in Circulating Progenitor Cells Due to Aldosterone Excess in Experimental Hypertension Barry A. Boilson1, Brian P. Shapiro1, Donna M. Meyer1, Adriana Harbuzariu1, Cheryl S. Mueske1, James E. Tarara1, Robert D. Simari1, Margaret M. Redfield1; 1 Division of Cardiovascular Diseases, Mayo Clinic College of Medicine, Rochester, MN Introduction: Bone marrow-derived progenitor cells may have a reparative and thus potentially therapeutic role in cardiovascular disease. Reduced reparative progenitor cell production or function may play a role in hypertension related vascular injury. Further, in vitro studies suggest that humoral activation (aldosterone) impairs production of progenitor cells, potentially limiting endogenous repair mechanisms and accelerating vascular damage during the transition to heart failure. Hypothesis: We hypothesized that circulating progenitor cells would be depleted in vivo in response to experimental hypertension and that these changes would be accelerated in the presence of concomitant aldosterone excess. Methods: Ten dogs underwent renal wrapping (RW) to induce hypertension. Blood pressure (BP) was measured weekly. After development of hypertension (5 weeks after RW), dogs were randomized to receive deoxycorticosterone acetate (1mg/kg/day for 3 weeks; HTN þ DOCA) or not (HTN). Flow cytometric analysis for CD34þ (progenitor) cells was performed on whole blood harvested prior to and every 2 weeks after RW. ISHAGE criteria were used to enumerate CD34þ cells. Results: See Figure. Dogs were similarly hypertensive at 5 weeks; BP increased week 5-8 only in HTN þ DOCA dogs. The CD34þ cell counts were similar prior to RW and decreased over time after RW in both groups. However, the decline in CD34þ cells was accelerated in the HTN þ DOCA dogs after DOCA treatment. Conclusion: This study demonstrates a progressive decrease in circulating progenitor cells over time after the onset of experimental hypertension. This may represent impaired bone marrow production or homing to sites of hypertension related vascular injury. However, the dramatic drop in CD34þ cells after DOCA administration is consistent with the previous in vitro studies suggesting that aldosterone impairs bone marrow derived progenitor cell formation. These data support the concept that humoral activation accelerates vascular injury by limiting endogenous reparative mechanisms.
095 Interferon Regulatory Factor-9 (IRF-9) Mediates Short Term Host Protection, but Promotes Long Term Immune Injury in Evolution of Myocarditis Leading to Dilated Cardiomyopathy Michael Konviser1,2, Youan Liu1, Manyin Chen1, Mei Sun1, Yu Shi1, Anthony Gramolini2, Anne Opavsky2, Eleanor Fish2, Peter Liu1,2,3; 1Toronto General Reasearch Institute; 2University of Toronto; 3Canadian Institute of Health Reasearch Introduction: Evolution of viral myocarditis to dilated cardiomyopathy (DCM) represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. However, the influence of innate immunity in general and IRF-9 in particular on acquired immunity and DCM is unknown. Hypothesis: IRF-9 signaling provides immediate host protection through interferon production, but stimulates acquired immunity leading to DCM in a coxsackievirus murine myocarditis model. Methods: Interferon-regulatory factor 9 (IRF-9) homozygous knockout mice were generated, and show impaired type I interferon production. Wild-type (WT, n 5 51) and IRF-9-/- (n 5 124) littermates were inoculated with 10^2 p.f.u. of coxsackievirus B3 as previously described. Survival, viral titers, cardiac hypertrophy, inflammation and fibrosis were evaluated on days 0, 4, 7, 10, 14, 28 and 42. Splenocyte subpopulations were cell sorted and quantitated by FACS. Results: IRF9-/- mice showed dramatically increased mortality compared to the wild-type littermates (0% WT vs 72% IRF-9-/- on day 14, P ! 0.0001). On day 42, there was less cardiac hypertrophy in IRF-9-/- mice compared to WT controls (p ! 0.05). There was no difference in inflammation and fibrosis. The mature T-lymphocyte population, defined as CD4 or CD8 single positive, was statistically identical in the two populations up until and including day 28 post-infection. However on day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9-/- spleens (p ! 0.05). Conclusions: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.
096 Identification of Genetic Variants of the Tumor Necrosis Factor Receptor 2 Gene (TNFRSF1B) in Patients with Heart Failure Akash Makkar1, Javier Banchs1, Mary A. Hauck2, Tariq S. Siddiqui1, Saeed A. Jortani3, Roland Valdes3, Steven C. Koenig2, Michael E. Mitchell4, Aoy TomitaMitchell4, Sumanth D. Prabhu1; 1Medicine, University of Louisville; 2Surgery, University of Louisville; 3Pathology, University of Louisville; 4Surgery, Medical College of Wisconsin Background: Tumor necrosis factor-alpha (TNF) plays an important pathophysiological role in heart failure (HF). As TNF signals exclusively via its two receptors (TNFR1 and TNFR2), TNFR polymorphisms may modulate the biological effects of TNF in HF and impact disease progression. The gene for TNFR2, TNFRSF1B, has been implicated through linkage studies in the pathogenesis of hypertension and coronary artery disease. However, whether TNFRSF1B is similarly linked to the pathogenesis of HF is unknown. Methods: To test the hypothesis that TNFRSF1B mutations contribute to HF, we conducted a pilot study in patients with systolic HF (LVEF ! 40%, NYHA class II-IV; excluded if recent [! 3 mo] infarction, significant valvular disease, or acute infection) to screen for mutations in the coding and flanking introns of TNFRSF1B and compared them to healthy controls without HF using heteroduplex analysis by capillary electrophoresis as well as direct sequencing. Results: We identified 3 previously described SNPs (K56K, M196R, E232K), 2 intronic mutations, and a novel synonymous mutation (A19A, patient with non-ischemic HF) in the coding region. The previously described SNPs occurred as haplotypes with the M196R SNP. The M196R SNP, previously reported to be associated with systemic lupus erythematosus (among other disorders), was found to occur at 51%