DNA Variants in NOS3 Are Associated with Improved Small Artery Elasticity and Decreased Systolic Blood Pressure in Asymptomatic Individuals

S34 Journal of Cardiac Failure Vol. 17 No. 8S August 2011 Fourteen miRNA transcripts were quantified following reverse transcription and realtime polymerase chain reaction; expression levels were normalized to an internal control. After testing for normality, group comparisons were performed using a ttest. ROC curves were generated to evaluate diagnostic utility of miRNA expression. Results: No age or gender association was observed with any miRNA expression. Of 14 miRNA tested, 6 were significantly associated with IS- HF, 5 with ID-HF and 4 with both (Table 1). By ROC analysis miRNA expression robustly differentiates HF groups from HC (Table 2). Table 1. MiRNAs Expression P value,HF/ ID/IS vs HC

miRNA

HC

HF

ID

IS

miR-1

7.1 6 4.3

14.4 6 5.1

14.6 6 6.0

14.3 6 4.5

miR-21 miR-126 miR-133a miR-133b miR-208 Let-7a

-1.1 -1.4 2.8 3.7 13.4 -3.0

6 6 6 6 6 6

2.9 2.7 2.8 2.8 3.1 2.7

0.9 0.3 4.6 1.6 15.1 -1.9

6 6 6 6 6 6

3.0 2.5 2.7 4.9 3.0 3.0

0.8 0.1 5.2 2.8 15.4 -2.6

6 6 6 6 6 6

3.3 2.7 2.7 4.9 2.7 3.6

1.04 0.4 4.2 0.8 14.8 -1.2

6 6 6 6 6 6

2.7 2.5 2.7 4.8 3.2 2.4

!0.000/!0.000/ !0.000 0.002/0.033/0.005 0.004/0.050/0.010 0.003/0.004/0.051 0.025/NS/0.015 0.014/0.019/NS NS/NS/0.010

Data expressed as a mean 6 standard deviation; miR, micro-RNA; HC, healthy control; HF, heart failure; IDC, idiopathic; CM, cardiomyopathy; ISC, ischemic. Table 2. MiRNA ROC Curve Analysis, AUC (p) miRNA miR-1 miR-21 miR-126 miR-133a miR-208 Let7a

HF 0.866 0.700 0.672 0.695 0.666 0.646

(!0.000) (!0.002) (!0.007) (!0.002) (!0.008) (!0.020)

ID CM 0.846 0.659 0.645 0.734 0.682 0.576

(!0.000) (0.059) (0.083) (0.005) (0.024) (0.348)

IS CM 0.881 0.730 0.692 0.666 0.652 0.706

(!0.000) (0.002) (0.011) (0.025) (0.046) (0.007)

All data expressed as area under the curve(AUC), c statistic; (p value). Conclusions: Several peripheral blood miRNAs are significantly disregulated in ID and IS patients when compared to normal controls. The potential use of these molecules or their combinations as a diagnostic tool deserves further exploration.

107 DNA Variants in NOS3 Are Associated with Improved Small Artery Elasticity and Decreased Systolic Blood Pressure in Asymptomatic Individuals Jennifer L. Hall1, Natalia Florez1, Marj Carlson1, Sara Saul1, Mark Lawson2, Aaron Mackey2, Joao Lima3, Jay Cohn1, Stephen S. Rich2, Wei Zhong1, Gregory Grandits1, Daniel A. Duprez1; 1Cardiology, Medicine, Lillehei Heart Institute, University of Minnesota, Minneapolis, MN; 2Center for Public Health Genomics, University of Virginia, Charlottesville, VA; 3Medicine, Johns Hopkins University, Baltimore, MD Small artery elasticity (SAE) is a functional marker that predicts the onset of arterial hypertension in normotensive subjects. The Multi-Ethnic Study of Atherosclerosis (MESA) showed that SAE is predictive of cardiovascular morbidity and mortality in subjects free of overt cardiovascular disease (CVD). SAE reflects endothelial (dys)function. The main objective was to determine if SAE and systolic blood pressure (SBP) were associated with DNA variants in the nitric oxide synthase 3 (endothelial, NOS3) gene. A total of 536 subjects (356 males, 180 females), free of overt CVD, who attended the primary CVD prevention clinic were enrolled in the study. SAE was derived from the diastolic pulse contour analysis of the radial artery waveform using non-invasive arterial tonometry. SBP was measured using an automatic blood pressure device. Nine tagging single nucleotide polymorphisms (SNPs) in NOS3 were genotyped using Sequenom. Logistic regression analysis was performed using PLINK software. The analyses modeled SAE with NOS3 SNPs and covariates (including age, race, and sex). Exome sequencing was performed in six subjects. In-solution exome capture was performed using Agilent SureSelect (37 Mb) and next-generation sequencing performed on Illumina Solexa GAIIx. Bioinformatic analyses were performed using available and custom pipelines at the University of Virginia. NOS3 SNP rs3730006 was associated with lower SBP (G/G, 129 +/- 17 mm Hg n5494, A/G, 125 +/- 14, n524, and A/A, 114 +/- 6, n52; p!0.05), and marginally associated with improved small artery elasticity (G/G 6.5 +/- 3.2, n5493, A/G, 6.9 +/- 3.8 +/- 3.8, n524, and A/A, 9.95 +/- 3.75, n 52; p ! 0.06). A significant association was found in a NOS3 haplotype block including SNPs rs1799983 (Glu298Asp) and rs3918227 with decreased SBP (p ! 0.05). Exome sequencing identified 40,720 SNPs; 39,174 of these had been previously identified and were in dbSNP. There were 1,546 SNPs that were novel. Of 40,720 SNPs, 93 represented missense/nonsense mutations. These novel findings suggest that DNA variants in NOS3 are associated with improved SAE and decreased SBP. Studies are on-going to validate missense/novel SNPs.

108 Oxidative Modification of the Extracellular Domain Decreases b1-Integrin Avidity and Interferes with Outside-In Signaling and Cell Survival Stephanie P. Hauselmann1, Vera Lorenz1, Otmar Pfister1,2, Gabriela M. Kuster1,2; 1 Department of Biomedicine, University Hospital, Basel, Switzerland; 2Division of Cardiology, University Hospital, Basel, Switzerland Background: We have previously shown that intracellular, Rac1/NADPH oxidasederived reactive oxygen species (ROS) regulate b1-integrin expression and mediate b1-integrin-induced outside-in pro-survival signaling in cardiomyocytes (CMC). b1-integrin exhibits a series of cysteines in the extracellular domain raising the possibility that oxidative thiol modification of these cysteine residues may alter b1-integrin function. Hypothesis: We sought to test the hypothesis that extracellular ROS participate in the regulation of b1-integrin function in cultured H9c2 cardiomyoblasts. Methods: H9c2 cells were treated with the membrane impermeant thiol oxidant 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB, 1-10mM) or the reductant glutathione (GSH, 1-10mM) and plated on culture dishes precoated with the b1-integrin ligand laminin (LN, 20mg/mL). Cell adhesion was determined by toluidine blue absorbance. Phosphorylation of focal adhesion kinase (FAK), extracellular signalregulated kinase (ERK) and Akt was analyzed by Western blotting. Cell viability was assessed by resazurin reduction. Results: LN induced a 3.9-fold increase in H9c2 cell adhesion after 1hr (p!0.001 vs non-coated dishes) and increased the phosphorylation of FAK, ERK and Akt. LN-induced cell adhesion was enhanced in GSH-treated cells, with peak adhesion reached at 2.5mM GSH (+4166%, p! 0.01). Conversely, DTNB dose-dependently inhibited the LN-induced cell adhesion, with maximal effect at 10mM (-4369%, p!0.001). Furthermore, DTNB also blunted the LN-induced FAK-, ERK- and Akt-phosphorylation and markedly reduced cell viability after 16hrs. Conclusions: Redox modulation of extracellular thiols interferes with b1-integrin avidity, thereby affecting cell adhesion, outside-in signaling and cell survival. Oxidative stress in the extracellular compartment thus may contribute to the disruption of cell-matrix interactions leading to detachment-induced cell death (anoikis), CMC slippage and ultimately ventricular dilation.

109 Right Ventricular Failure in Patients Requiring Mechanical Circulatory Assistance Is Associated with Elevated miR-15b Expression Alexander G. Moore1, Marc Simon2, Bonnie Lemster2, John Gorcsan2, Charles McTiernan2, Ravi Ramani2; 1University of Pittsburgh School of Medicine, Pittsburgh, PA; 2UPMC Cardiovascular Institute, Pittsburgh, PA Introduction: Right ventricular (RV) failure imposes a markedly worse prognosis and quality of life in patients with heart failure, particularly in those requiring mechanical circulatory support. MicroRNAs (miRs), through their contribution to gene regulation, have been implicated in the pathogenesis of heart failure and have demonstrated promise as biomarkers for clinically significant events. Their role in right ventricular failure, however, is unknown. Hypothesis: Patients with severe ventricular dysfunction requiring biventricular mechanical support (BiVADs) will demonstrate a myocardial miR expression profile distinct from otherwise similar patients who require only left ventricular support (LVAD). Methods: Left ventricle apical core samples collected at the time of ventricular assist device (VAD) implantation were analyzed from two groups: patients requiring BiVADs (n55) and clinically matched patients requiring only LVADs (n55). Patients were matched according to etiology of heart failure, sex, left ventricle size, cardiac output, and right heart and pulmonary pressures. RNA extracted from these tissue samples was assayed for the expression of seven miRs previously shown to play a role in heart failure using quantitative RT PCR. miRs analyzed included miRs-1, 15b, 21, 23a, 133a, 133b, and 195. Data was normalized to expression of the small RNA RNU48. Results: miR-15b was expressed at a significantly higher level in patients requiring biventricular assistance (1.33 fold expression (p! .04)) compared to patients requiring only left ventricular assistance. The relative expressions of miRs-1, 21, 23a, 133a, 133b, and 195 were not found to be significantly different. Conclusions: The need for mechanical right ventricular support is associated with elevated expression of miR-15b. miRs may be useful markers of right heart failure in patients receiving VADs and may provide mechanistic insights into the biology of biventricular dysfunction through the identification of transcripts regulated by miR-15b.

110 Right Ventricular Metabolic Imaging in Experimental Pulmonary Artery Hypertension Lisa M. Mielniczuk, Stephanie Thorn, Jennifer J. Renaud, Ran Klein, Jean DaSilva, Robert J. deKemp, Rob A. Beanlands, Duncan J. Stewart; Cardiology, University of Ottawa Heart Institute, Ottawa, ON, Canada Background: Pulmonary artery hypertension (PAH) is a disease of progressive vascular remodeling, vasoconstriction, and right heart failure (HF). There is heterogeneity in the development of right HF, and the mechanisms and predictors remain largely unknown. It has been suggested that alterations in cardiac metabolism may be related to progressive RV dysfunction. This study was designed to evaluate the changes in fatty acid and glucose metabolism with cardiac PET imaging in experimental PAH. Methods/Results: Monocrotaline was given as