128Autologous Bone Marrow Mononuclear Cell Transplantation (ABMMCT) forPatients with Severe Ischemic Heart Failure: Correlation of the CellsPhenotypes with the Analyzed Clinical VariablesSuzana A. Silva1, Maria Izabel D. Rossi2, Radovan Borojevic2, Andre L. Sousa1,Guilherme V. Silva3, Emerson C. Perin3, Hans F. Dohmann1; 1Research andTeaching Centre of Pro Cardiaco, Hospital Pro Cardiaco/PROCEP, Rio de Janeiro,Brazil; 2Department of Histology and Embryology, University Federal of Rio deJaneiro, Rio de Janeiro, Brazil; 3Intervention Cardiology, Texas Heart Institute,Houston

Objective: This study aims to correlate the different cells phenotypes of the totalpool of BMMC injected in severe ischemic heart failure pts with the 1 year improve-ment of analyzed clinical variables. Methods: 21 pts were enrolled in this prospec-tive study (first 14 pts treated group (TG) and the last 7 pts control group (CG)).Initially all pts underwent clinical and laboratorial assessment, SF-36 e Minnessotaquestionnaire, ramp treadmill, echocardiogram, scintigraphy, SA-ECG e 24h Holter.BMMC were injected using MyoStar� NOGA catheter. Before procedure, TG under-went to initial angiography followed by electromechanical mapping. Non invasivetests were reassessed after 2; 6 and 12 mths of FU in all pts. Angiography and elec-tromechanical mapping were repeated only in TG at 4 months. The relative improve-ment of these clinical variables was correlated to %, total number and density ofBMMC injected and their different phenotypes, through Spearman’s rho test (p #

0.05). Cells phenotypes of pts with reversible defect sustained improvement werecompared to the pts without this favorable evolution. Results: Demographics didnot differ between groups. Clinical results were previously reported. The viable cellsand the cells fenotype CD45lo, HLADR-, ckitþCD45-, CD14þ, CD19þ, CD34þ,CFU-F and CFU-GM were positively correlated to the clinical improvement of ana-lyzed clinical variables, while the cells CD56þ were negatively correlated to it. TheCD4þ, CD8þ and CD19- cells were ambiguously correlated to those variables. Ptswith sustained improvement of myocardial ischemia had a higher number and densityof CFU-GM (p!0,03) and CD34þHLADR- (p!0.05) as well as lower number ofCD19þ (p 5 0.01), CD4þ (p 5 0.05) and CD8þ (p 5 0.07). Conclusion: This studyshowed that ABMMCT is safe and feasible. There was a significant correlationamong the cells and analysed clinical variables. There was a higher number and den-sity of viable cells, CD34þ and CFU-GM as well as a lower number and density ofCD4þ, CD8þ and CD19þ in pts with 1 year sustained improvement of ischemia. Itis possible that these cells maintain a long term paracrine effect on myocardium.

129Generation of a Transgenic (Tg) Mouse Model with Cardiac SpecificOverexpression of a Dominant Negative (DN) Mutant of p67phox

Lei Xiao1, Yanmei Du1, Guangjie Cheng2, J. David Lambeth2, Roberta Franks3,Qiong Zhao1; 1Center for Cardiovascular Research, University of Illinois atChicago, Chicago, IL; 2Pathology and Laboratory Medicine, Emory University,Atlanta, GA; 3Transgenic Production Service, University of Illinois at Chicago,Chicago, IL

NADPH oxidase (NOX) has been shown to play a critical role in the development ofcardiac hypertrophy induced by angiotensin II and a1-adrenergic receptor (AR) stim-ulation. Overexpression of a V204A DN mutant of NOX cytosolic subunit p67phox

(DN-p67) was reported to prevent NOX-mediated superoxide generation in vitro. Re-cently, we found that overexpression of the DN-p67 mutant attenuated a1-AR-in-duced hypertrophy in cultured cardiac myocytes. In order to further investigate therole of NOX/67phox in cardiac hypertrophy in vivo, we generated a Tg mouse modelwith cardiac-specific overexpression of the above V204A DN-p67 coding region. Forthe convenience of transgene screening, an enhanced green fluorescent protein(EGFP) gene was linked downstream to the DN-p67 transgene. The independenttranslation of the EGFP was initiated by a leading viral sequence for internalribosome entry site (IRES) that was linked to the 3’end of DN-p67. The DN-p67-IRES-EGFP transgene was next linked to the 3’end of a cardiac-specifica-MHC promoter generating a 9.1-Kb transgene that contains the a-MHC promoter,the DN-p67 coding region, the IRES-EGFP sequence, and a polyadenylation signalsequence. This new transgene was then injected into fertilized C57BL/6J zygotesthat were transplanted into pseudopregnant mice. The obtained offspring micewere screened for transgene integration by PCR analysis and Southern blotting oftail DNA. Two pairs of PCR primers were used in screening to detect eithera 640-bp EGFP coding sequence or a 277-bp region span the junction between thea-MHC promoter and the DN-p67 coding sequence. Three Tg-positive foundermice (2 male and 1 female) were obtained. Currently, at age of 18 weeks, the 3founder mice are all viable and healthy with normal phenotypes. At age of 10 weeks,each of the 3 founders was bred to wild type (WT) C57BL/6J mice independently,and had produced 6 DN-p67 Tg offspring at present. These heterozygous Tg litter-mates will be used to independently breed for homozygous DN-p67 Tg lines derivedfrom each of the 3 Tg founders. In summary, we have generated a Tg mouse modelwith cardiac-specific overexpression of a DN mutant of p67phox, which would bea useful in vivo model for studying cardiac diseases involving NOX/p67phox.

S40 Journal of Cardiac Failure Vol. 12 No. 6 Suppl. 2006

130GRK2 Expression in Lymphocytes of Human Patients with Heart Failure-ANew BiomarkerAmit Mittal, Noah Bloomgarden, J. Kurt Chuprun, Jeffrey Martini, Brent R.DeGeorge, Jr., Sivakumar Srinivasan, Raphael Bonita, Andrea D. Eckhart, Paul J.Mather, Walter J. Koch; Center for Translational Medicine, Thomas JeffersonUniversity, Philadelphia, PA; Advanced Heart Failure and Cardiac TransplantCenter, Thomas Jefferson University Hospital, Philadelphia, PA

Introduction: Despite numerous therapeutic advances recently for heart failure (HF),hospitalizations and deaths are still rising. Thus, new treatments as well as improvedbiomarkers are critically needed to improve the overall care of HF patients. Recently,we have shown that G-protein Coupled Receptor Kinase-2 (GRK2) expression in pe-ripheral lymphocytes tracks with myocardial levels and higher levels in the blood isassociated with more severe HF. Hypothesis and Methods: Our hypothesis is thatblood GRK2 is a novel biomarker in HF that may help represent a surrogate marker.In order to solidify our previous results showing that lymphocyte GRK2 is negativelyassociated with LV function in HF, we need to compare levels in HF patients to nor-mal control subjects without LV dysfunction. We have determined lymphocyte GRK2protein levels in HF and control subjects in this study. HF subjects were recruited andconsented during their hospitalization or their outpatient visit to a HF specialist.Patients with normal LV function and taking no cardiovascular medicines were alsorecruited. Blood was drawn from these subjects and the white blood cells were isolatedusing Ficoll gradient centrifugation. Purified lymphocyte protein extracts were used forGRK2 protein immunoblotting and quantification via densitometry and normalizationwith the housekeeping protein, GAPDH. Results: GRK2 levels expressed as a ratio be-tween GRK2 expression and GAPDH (GRK2/GAPDH) in the normal control group(n 5 30) was 0.264 6 0.068. Importantly, in HF patients (n 5 20), we found a significant3-4 fold increase in GRK2 levels in peripheral blood, 1.00 6 0.258 GRK2/GAPDH(P!0.05). Moreover, there was a relationship between GRK2 levels and disease sever-ity (HF class) in the HF patients. Conclusions: With a larger number of study subjects,we have confirmed that GRK2 in white blood cells is elevated in human patients withfailing hearts when compared to subjects with normal LV function. Future studies willcompare GRK2 levels in HF patients treated with standard pharmacologic and devicetherapy to test our hypothesis that GRK2 can be a surrogate marker for determination ofa given patient’s response to treatment.