C-kit–Positive Cardiac Progenitor Cells Contribute to the Development of the Mammalian Heart

Topics Include: • Fundamental Mechanisms of Cardiac Regeneration • Signaling in Vascular Smooth Muscle and the Heart • Calcium Handling in Cardiac Muscle by gest on Sptem er 4, 2017 http://circhajournals.org/ D ow nladed from Emerging Concepts in Cardiovascular Biology I: Late-Breaking Basic Science Oral Abstracts C-kit–Positive Cardiac Progenitor Cells Contribute to the Development of the Mammalian Heart Grazia Iaffaldano, Yu Misao, NEW YORK Med COLLEGE, Valhalla, NY; Laura Perin, Childrens Hosp Los Angeles, Los Angeles, CA; Luca Melchiori, Michael Arcarese, Narissa Small, Marcello Rota, NEW YORK Med COLLEGE, Valhalla, NY; Leonard G Meggs, Univ of Medicine and Dentistry New Jersey, Newark, NJ; Annarosa Leri, NEW YORK Med COLLEGE, Valhalla, NY; Roberto Bolli, Univ of Louisville, Louisville, KY; Sergio Ottolenghi, Univ of Milan, Milan, Italy; Mark A Sussman, San Diego State Univ, San Diego, CA; Piero Anversa, Jan Kajstura; NEW YORK Med COLLEGE, Valhalla, NY A population of c-kit-positive cardiac progenitor cells (CPCs) has been identified in the adult mammalian heart and these cells function as stem cells in vitro and in vivo. The aim of this study was to determine whether c-kit-CPCs are present during embryonic development and participate in the formation of the four-chambered heart. For this purpose, a transgenic mouse in which EGFP is regulated by the c-kit promoter was developed to characterize the presence and anatomical distribution of this class of progenitor cells within the forming myocardium. Moreover, the migration of these cells from other organs to the heart was established to define the origin of the c-kit-CPC pool. Embryo culture was employed from E7 to E13 and organ culture was used from E14 to E19. These preparations of live embryo and heart were followed by two-photon microscopy for a period of 5–8 hours. Subsequently, embryos and hearts were fixed and whole-mount immunostaining was utilized for further analysis. Nearly 20 green fluorescent cells were detected at E8.5 and the number of these cells increased nearly 10-fold at E11.5. The expression of c-kit in the EGFP positive cells was confirmed by immunolabeling. EGFP-positive-c-kit-positive-cells were present in other regions of the embryo but never translocated to the myocardium during the 5–8 hours of observation. A further 1.6-fold increase in the number of c-kit-CPCs was found at E15. At E13-E15, clusters of c-kit-CPCs were detected at the apex and later throughout the ventricles and atria. C-kit-CPCs exhibited morphogenic movements and concurrently divided. Cytokinesis occurred in less than 2 hours. The c-kit-CPCs were nested within the growing myocardium where they formed junctional complexes with the neighboring cells by the expression of connexin and cadherin. Importantly, a relevant fraction of c-kit-CPCs acquired myocyte characteristics. Although these immature cells retained partly c-kit, Nkx2.5 was identified in the nuclei and -actinin was localized in the cytoplasm. These properties indicate that the c-kit-CPCs were capable of giving rise to a myocyte progeny. In conclusion, cardiac development is regulated in part by resident c-kit-CPCs which are implicated in the generation of the four-chambered heart. Do Cardiac Stem Cells Arise From Cardiomyocyte Dedifferentiation? Yiqiang Zhang, Kuan-Cheng Chang, Carmen C Delgado, Ting Liu, Rachel Smith, Roselle M Abraham, Mark Pittenger, Eduardo Marbán; JOHNS HOPKINS UNIV, Baltimore, MD Background: Working cardiomyocytes have long been considered to be terminallydifferentiated. Studies in amphibians and zebrafish have demonstrated the capability of adult cardiomyocytes to re-enter the cell cycle and repair the injured heart. Objectives: 1) We assessed the ability ofasked whether adult mammalian cardiomyocytes tocould re-enter the cell cycle, proliferate and express stem cell markers after dedifferentiation. 2) We characterized the ionic remodeling during cardiomyocyte dedifferentiation. Methods and Results: Adult atrial cardiomyocytes isolated from guinea pigs or rats were cultured in mitogen-rich media. Cells were plated sparsely on grid-marked cover slips and followed by time-lapse video microscopy. Myocytes started to dedifferentiate and to lose their striated pattern after culturing for 2 to 4 days. Such cells beat spontaneously and gave rise to new cells, as identified by serial inspection on numbered grids. We found that the inwardly-rectifying potassium current and the resting membrane potential were reduced significantly in dedifferentiating myocytes and in newly-divided cells. Immunocytochemistry (ICC) and confocal laser scanning microscopy revealed that cardiomyocytes re-entered the cell cycle, at least partially due to the diminished expression of 14–3–3 which is an important cell cycle regulator. With prolonged culture, myocytes produced small phase-bright cells that were positive for stem cell markers; 67% of these phase-bright cells expressed c-kit and 54% expressed Sca-1.. Conclusion: Myocyte dedifferentiation may serve as a source for cardiac regeneration. A Novel Method to Commit Mesenchymal Stem Cells to a Cardiac Lineage Results in Improved Mechanical Function and MSC-Derived Striated Cardiac Myocytes in the Adult Canine Heart Irina A Potapova, Sergey V Doronin, Damon J Kelly, Amy B Rosen, Adam J Schuldt, Yuaniian Guo, Zhongju Lu, SUNY Stony Brook, Stony Brook, NY; Richard B Robinson, Michael R Rosen, Columbia Univ, New York, NY; Peter R Brink, SUNY Stony Brook, Stony Brook, NY; Glenn Gaudette, Worcester Polytechnic Institute, Worcester, MA; Ira S Cohen; SUNY Stony Brook, Stony Brook, NY Introduction: Whether human mesenchymal stem cells (hMSCs) can differentiate into functional cardiac myocytes is still controversial. However, embryonic stem cells are known to differentiate into myocytes through the formation of embryoid bodies. We now demonstrate commitment of adult hMSCs to a cardiac lineage through the use of embryoid bodies. Methods: Cardiogenic cells were produced by forming embryoid bodies with hMSCs. We used immunocytochemistry to identify cardiac-specific markers and patch clamping to measure Ca currents. To determine the ability of these cells to provide mechanical function in vivo, cardiogenic cells were incubated on an acellular scaffold for 3– 8 days and then implanted in a full thickness ventricular defect in canine hearts. Un-manipulated hMSCs were a control. Cardiogenic cells were loaded with quantum dots (QD) to track their fate. Results: Cardiogenic cells, but NOT un-manipulated hMSCs, expressed cardiac specific markers alpha sarcomeric actinin, atrial natriuretic polypeptide, cardiotin, troponin T, and Cav1.2 (the alpha subunit of the L-type calcium channel). In some cells, actinin and troponin T staining showed myofibrillar organization. A majority of cardiogenic cells expressed cyclin D1 in the nucleus. L-type calcium currents (-9.3 1.3 pA/pF at 10 mV; n 5) were recorded in some cardiogenic cells of size similar to adult cardiac myocytes. When implanted in the heart, compared to un-manipulated hMSCseeded patches (n 5), cardiogenic cell seeded patches (n 8) improved normalized regional stroke work (7 0.8 vs. 4 0.5; p 0.05) and systolic contraction (8 1.0% vs. 4 1.2%; p 0.05). Also, QD positive cells with sarcomeres were found in the implantation area, suggesting that cardiogenic cells differentiated into striated cardiac myocytes.Conclusions: We have developed a method for rapid commitment of hMSCs to a cardiac lineage. The cells express cardiac specific markers, maintain proliferative capacity and some have robust calcium currents. In vivo, these cells significantly improve mechanical function compared to control hMSCs. In addition, 8 weeks after implantation, these cells possess sarcomeres. These cardiogenic cells are a potential new cellular source for mammalian myocardial regeneration. Mobilized Peripheral Blood Contains a Very Rare Population of Pluripotent Mesenchimal Stem Cells Able to Differentiate Into Myocytes Silvia Rigo, Antonio Paolo Beltrami, Daniela Cesselli, Natascha Bergamin, Patrizia Marcon, Elisa Puppato, Federica D’Aurizio, Alessia Coletto, Daniela Damiani, Renato Fanin, Carlo Alberto Beltrami; CIME (InterDeptal Cntr of regenerative medicine) Univ OF UDINE, Udine, Italy Experimental studies based on the use of granulocyte colony-forming factor (GCSF) raised new hope for the myocardial infarction treatment; however, GCSF-based clinical trial outcomes were quite contradictory and disappointing. Recently, we have demonstrated the existence of a class of Pluripotent Mesenchymal Stem Cells (PMSCs) residing in heart, bone marrow and other human tissues and therefore we decided to investigate whether PMSCs can be isolated both from non mobilized and GCSF-mobilized peripheral blood. Aims: to verify if: 1. Conventional Hematopoietic Stem Cell (HSC) mobilizing regimens are able to recruit PMSCs into peripheral blood (PB); 2. PB-derived PMSCs can be expanded in vitro and differentiate towards a myogenic fate. Materials and Methods: Buffy coats from GCSF-mobilized healty donors (N 8) and non-mobilized blood donors (n 14) were cultured for 8 days in a high serum medium and then transferred to a low serum medium containing PDGF-BB and EGF. Results: We failed in generating proliferating PMSC lines from non-mobilized PB, while we succeeded from 2 out of 8 mobilized PB. Growing cells could be expanded up to 70 population doublings without reaching senescence, as testified by the absence of p16, p21 and p53 expression as well as telomere dysfunction foci. Cells displayed a mesenchymal immunophenotype and expressed the Pluripotent State Specific Transcription Factors OCT-4 and Nanog, both at protein (83% and 62.5%, respectively) and at mRNA levels. When cultured in differentiation-inducing conditions, they were able to generate functionally competent multiple derivatives of the three germ cell layers. In particular, in a myogenic medium 70% of the cells expressed organized alphasarcomeric actin and connexin-43 and presented spontaneous intracellular calcium transients. Conclusions: This is the first time that PMSCs from human PB have been isolated, expanded in vitro and differentiated into functionally competent myocytes. The low percentage of subjects displaying growing PB-derived PMSCs, even after GCSF treatment, can be considered as a consequence of different individual mobilization capacities and reveals as conventional HSC-mobilization regimens may be not effective in mobilizing or activating this class of stem cells. Vascular Side Population Cells May Participate in Aging and Vascular Pathology Via Regulation of Their ABCG2-Membrane Transporter Julie Sainz, Masataka Sata, Takaaki Hasegawa, Ibuki Shirakawa, Yumi Sugawara, Hiromi Kato, Ryozo Nagai; Dept of Cardiovascular Medicine, Univ of Tokyo, Graduate Sch of Medicine, Tokyo, Japan Objectives: We previously identified Side Population (SP) progenitor cells expressing the ABCG2 stem cell-marker in normal murine arteries. To determine whether they could be involved in development of vascular diseases, we compared the variation of the SP in normal and atherosclerotic arteries, according to age. Moreover, we evaluated the role of ABCG2 in SP cell proliferation and neointima development following vascular injury, in vitro and in vivo. Methods and Results: Arterial SP cells were found in similar proportion in 8 week-old wild type (4.8 0.4%) and ApoE (3.9 1.0%) mice. However, the SP highly increased with aging in wild type mice (13.6 1.7% at 74 weeks; 20.2 1.1% at 131 weeks), and this increase was significantly more dramatic in ApoE mice (34.1 3.1% at 74 weeks, p 0.005). Interestingly, in vitro BrdU assay showed that SP cell proliferation was enhanced two to three-fold in the presence of PDGF-BB (10ng/ml), a high-mitogenic growth factor expressed in atherosclerotic arteries, and that this increase could be diminished by adding FumitremorginC, a specific ABCG2-inhibitor. To further investigate the role of ABCG2 in arterial response to injury in vivo, we placed a perivascular-cuff on common carotid and femoral arteries of wild type and ABCG2 mice. Four weeks later, animals were sacrificed and the neointima/media area ratio quantified. The ratio was significantly lower in carotid arteries (p 0.01) and femoral arteries (p 0.05) of ABCG2 mice, compared to that of wild type. Conclusions: SP progenitor cell prevalence increases during physiological aging and atherosclerosis development. Consistent with these results, mice which do not express ABCG2, an SP-marker, develop less neointima following vascular injury than wild type mice. These findings suggest that SP cells, which 1278 Circulation Research Vol 99, No 11 November 24, 2006 Basic Science by gest on Sptem er 4, 2017 http://circhajournals.org/ D ow nladed from proliferation may be regulated by the ABCG2-membrane transporter, could play a role in vascular pathology. Loss-of-Function Mutations in the Cardiac Calcium Channel Underline a New Clinical Entity Characterized by ST Segment Elevation, Short QT Intervals, and Sudden Cardiac Death Charles Antzelevitch, Guido D Pollevick, Jonathan M Cordeiro, Masonic Med Rsch Laboratory, Utica, NY; Oscar Casis, CVRTI, Univ of Utah, Salt Lake City,Utica, UT; Michael C Sanguinetti, CVRTI, Univ of Utah, Salt Lake City, UT; Yoshiyasu Aizawa, Alejandra Guerchicoff, Ryan Pfeiffer, Antonio Oliva, Masonic Med Rsch Laboratory, Utica, NY; Bernd Wollnik, Cntr for Molecular Medicine Cologne, Institute of Human Genetics Univ of Cologne, Cologne, Germany; Philip Gelber, Elias P Bonaros, Jr, Cardiovascular Consultants of Long Island, New Hyde Park, NY; Elena Burashnikov, Yuesheng Wu, Masonic Med Rsch Laboratory, Utica, NY; John D Sargent, CVRTI, Univ of Utah, Salt Lake City, UT; Stefan Schickel, Ralf Oberheiden, Dept of Internal Medicine, Academic Hosp Oberhausen, Oberhausen, Germany; Atul Bhatia, Univ of Wisconsin Med Sch, Milwaukee, WI; Li-Fern Hsu, Michel Haissaguerre, Hopital Cardiologique du Haut-Leveque, Bordeux, France; Rainer Schimpf, Martin Borggrefe, Christian Wolpert; 1st Dept of Medicine-Cardiology, Univ Hosp Mannheim, Faculty of Clinical Medicine of the Univ of Heidelberg, Mannheim, Germany Background: Cardiac ion channelopathies are responsible for an ever-increasing number and diversity of familial cardiac arrhythmia syndromes. We describe a new clinical entity consisting of an ST segment elevation in the right precordial ECG leads, a shorter than normal QT interval and a history of sudden cardiac death (SCD). Methods: Genomic DNA was prepared from peripheral blood lymphocytes. All known exons of 13 candidate genes were amplified using intronic primers and sequenced. Site-directed mutagenesis was performed and CHO-K1 cells were co-transfected using a 1:1:1 molar ratio of WT or mutant CACNB2b (Cav 2b), CACNA2D1 (Cav 2 1) and CACNA1C tagged with enhanced yellow fluorescent protein (Cav1.2). Whole-cell patch clamp studies were performed after 48 –72 hours of incubation. Results: QTc ranged from 330 to 370 ms in probands and clinically affected members of the family. Adaptation of QT interval to heart rate was reduced and in some cases tall peaked T waves were observed in the ECG. Quinidine normalized the QT interval and prevented programmed electrical stimulation induction of ventricular tachycardia. Genetic and heterologous expression studies revealed loss of function missense mutations in CACNA1C (A39V and G490R) and CACNB2 (S481L) encoding the 1 and subunits of the cardiac L-type calcium channel. Confocal microscopy showed that loss of current observed with the A39V mutation in Cav1.2 is due to a defect in trafficking of mature Cav1.2 channels from the ER/Golgi complex to the cell membrane, whereas channels formed from G490R Cav1.2 or S481L Cav 2b subunits traffic normally. Conclusion: This is the first report of loss of function mutations in two separate genes encoding different subunits of the cardiac L-type calcium channel to be associated with a familial SCD syndrome in which a Brugada syndrome phenotype is combined with a shorter than normal QT interval. Cardiac Resynchronization Therapy Improves Beta Adrenergic Reserve Response Through a RGS-Mediated Mechanism Khalid Chakir, Samantapudi Daya, Richard Tunin, David A Kass; Johns Hopkins Univ, Baltimore, MD Background Cardiac resynchronization therapy (CRT) improves symptoms and mortality in patients with left bundle branch (LBB) block and heart failure. Whether CRT reverses myocyte abnormalities associated with dyssynchronous heart failure (DHF) is unknown. Here, we tested if CRT improves basal and/or beta-adrenergic receptor ( -AR) stimulated function of isolated cardio-myocytes. Methods Adult dogs (n 12) underwent LBB radio-frequency ablation; half were subjected to right atrial tachypacing-induced DHF for 6 weeks, while the other half, DHF was induced in the first 3 weeks and followed by 3 weeks of CRT. An additional 6 non-paced dogs served as controls. Myocytes were enzymatically isolated from the anterior LV free wall and field stimulated in a perfusion chamber. Sarcomere shortening was determined with and without 1, 2 (or both) agonists. Results: DHF and CRT hearts had similar global failure, with reduced EF (28.4 8.1% vs 24.0 2.6%), dP/dtmx (1190.2 139.8 vs 1195.3 137.6 mmHg/s) and elevated LV end-diastolic pressure (35.7 3.7 vs 32.5 4.6, mmHg), respectively. However, there were marked differences in basal and -stimulated myocyte responses (Table, n 8 –30 cells/group). DHF myocytes showed depressed basal and -AR stimulation versus control. In contrast, myocytes from CRT hearts had enhanced basal and -AR stimulated function. Pretreatment of DHF myocytes with pertussis toxin (1.5 ug/ml for 3 hrs) improved -AR responses, supporting a role of enhanced Gi-AR coupling. RGS2 and RGS3 expression were greater in CRT than DHF (p 0.01 and p 0.0001 respectively), potentially explaining this difference. However, RGS4 did not show significant expression differences between DHF and CRT. Conclusion: CRT induces marked improvement in basal and AR-reserve myocyte function in part by inhibiting -AR/Gi coupling. This occurred despite persistent global failure, suggesting an effect from CRT per se, and may help explain improved functional capacity from CRT. Stimulation Protocol Sarcomere Fractional Shortening (%) Control DHF CRT DHF PTX Baseline 5.2 0.4 2.8 0.2* 5.2 0.4 3.2 0.7* 1 and 2 (ISO 10-7M) 14.2 0.5† 5.8 0.5†‡ 11.6 1.0† ‡ 8.4 0.6† ‡ 1 (NE 10-7 Prazosin 10-6) 11.5 0.8† 5.9 0.5†‡ 9.6 1.0† ‡ ND 2 (ISO 5*10-7 CGP 3 10-7) 10.4 0.5† 5.1 0.5†‡ 7.9 0.8† ‡ ND ISO isoproterenol, NE Norepinephrine, ND not done *; p 0.05 vs baseline control and baseline CRT , †; p 0.001 vs baseline, ‡, p 0.001 vs. Control, , p 0.01 vs. DHF, , p 0.01 vs DHF PTX, A MicroRNA Encoded by the beta-Myosin Heavy Chain Promoter Is Associated With Cardiomyopathic Alterations Patrick K Umeda, Regina P Shiau, James B Caulfield; Univ Of Alabama @ Birmingham, Birmingham, AL Transgenic mice containing sequences of the rabbit myosin heavy chain gene promoter exhibit many of the structural and functional alterations of heart failure. These phenotypic changes such as diminished contractile function, altered calcium homeostasis and an excess of mitochondria are mediated by an antisense RNA transcribed from the promoter sequences. To elucidate the mechanism by which this antisense RNA mediates its effect, we have characterized the structure of the antisense RNA by amplifying (by PCR) and cloning of its terminal sequences. The analyses indicates transcription of the antisense RNA occurs at approximately 160 base pairs upstream of the capsite of the myosin heavy chain gene and extends throughout the length of the transgene promoter sequences. The data confirms previous deletion analyses of the promoter in transgenic mice and also reverse transcription-PCR analyses of the antisense transcript. In some transgenic lines, the antisense RNA terminates following a cryptic polyadenylation signal in the adjoining gene sequences. However, there are no extensive open reading frames within the antisense RNA to suggest that it encodes a protein. The analyses also identified a small RNA of 21 nucleotides (nt) that is encoded by the rabbit promoter and derived from the antisense RNA transcript. Sequences of this 21-nt RNA can form a hairpin structure with adjacent sequences suggesting that it corresponds to a microRNA. MicroRNAs modulate gene expression by interacting with 3’-untranslated mRNA sequences and inhibiting the translation of the selected mRNA. Using the algorithm, MIRANDA, we used the sequence of the 21-nt RNA to identify potential target mRNAs. Selecting only target sequences that are highly conserved in human, mouse, and rat mRNAs, we identified approximately 20 potential target genes. The list includes genes such as transcription factors, splicing factors, signal transduction proteins and ion transporters. A number of these genes have been implicated in cardiomyopathy and heart failure. The results suggest that a microRNA gene encoded by the myosin heavy chain promoter may play an important role in mediating cardiomyopathic alterations in the failing heart. KCNA5 Loss-of-Function Mutation Implicates Tyrosine Kinase Signaling in Human Atrial Fibrillation Dawood Darbar, Tao Yang, Ping Yang, Gayle Kucera, Tanya Stubblefield, Jude McElroy, Dan M Roden; Vanderbilt Univ Med Schl, Nashville, TN Background: Atrial fibrillation (AF) is clinically-heterogeneous with risk factors that include family history. To identify genetic defects conferring disease susceptibility, we screened the voltage-gated cardiac potassium channel KCNA5 (underlying IKur), that is preferentially expressed in human atria, in 104 patients with idiopathic (lone) AF. Methods and Results: A 33-bp coding region deletion that has not previously been reported was identified in 2 Caucasian probands. One proband was part of a kindred that included 3 other family members with AF or palpitations, and all 4 were mutation carriers; no family was available in the 2 proband. The mutation results in deletion of 11 amino acids at positions 71–81 in the N-terminus of the protein, a proline-rich region previously implicated as a binding site for Src homology 3 (SH3) domains associated with Src-family protein tyrosine kinase (tk)-mediated phosphorylation and suppression of KCNA5 current. In transfected CHO cells, the mutant resulted in 60% decreased IKur vs wild-type (wt) (75 8 vs 180 15 pA/pF, after 500-msec pulses at 50 mV, n 14 each, P 0.01) and dominant-negative suppression of wt current (105 10 pA/pF, n 12–14, P 0.01 vs wt). Acute or chronic exposure to a v-Src tk suppressed the wt current by 90% and pretreatment with the Src inhibitor PP2 (10 M) prevented this Src-induced current suppression. By contrast, the mutant channel displayed no response to acute or chronic v-Src tk. Conclusions: We have identified a novel KCNA5 mutation in familial AF. This indel disrupts a proline-rich motif involved in tyrosine kinase regulation of IKur, decreases unstimulated current, but renders the channel kinase-resistant. These data implicate abnormal atrial action potential control due to variable tyrosine kinase signaling as a mechanism in this familial form of AF, and thereby suggest a role for modulation of this pathway in AF and its treatment. SREBP1 Repairs the Parasympathetic Dysfunction in Atrial Myocytes From Diabetic Hearts Ho-Jin Park, Yali Zhang, Chuang Du, Mark Aronovitz, Serban P Georgescu, Isaac Naggar, Christopher Madias, Tufts New England Med Ctr, Boston, MA; Charles M Welzig, Med Univ of South Carolina, Charleston, SC; Clayton E Mathews, The Univ of Pittsburgh, Pittsburgh, PA; Bo Wang, Ronglih Liao, Brigham & Women’s Hosp, Boston, MA; Robert O Blaustein, Richard H Karas, Jonas B Galper; Tufts New England Med Ctr, Boston, MA Parasympathetic dysfunction associated with Diabetic Autonomic Neuropathy has been implicated in the pathogenesis of sudden cardiac death in diabetics. We have previously shown that the diabetic Akita mouse which has a point mutation in the ins2 gene demonstrates a markedly reduced negative chronotropic response of the heart to parasympathetic stimulation. The parasympathetic response of the heart is mediated by the inward rectifying K current, IKAch, via acetylcholine activation of the G-protein coupled inward rectifying K channel (GIRK1/4). Here we describe a new molecular mechanism for parasympathetic dysfunction in the diabetic heart. In cultured atrial myocytes, insulin increased the expression of Sterol Regulatory Element Binding Protein (SREBP1), a transcription factor which plays a major role in lipid homeostasis, by 1.8 0.4 fold (N 3) and the expression of GIRK1 by 2.4 0.5 fold (N 5). Adenoviral expression of a DN-SREBP abolished insulin stimulation of GIRK1 expression in these cells, suggesting that SREBP regulates GIRK1 expression. Conversely, western blot analysis of extracts of hearts of hypoinsulinemic diabetic mice demonstrated a marked decrease in SREBP1 levels compared to WT. Atrial myocytes acutely dissociated from adult diabetic mouse hearts exhibited a decrease in carbamylcholine stimulation of IKAch with a peak value of -181 31 pA/pF (N 5) compared to -451 62 pA/pF (N 5, p 0.005) in cells from WT mice. Further, western blot analysis of extracts from atria of diabetic Akita mice Basic Science Late Breaking Abstracts 1279 by gest on Sptem er 4, 2017 http://circhajournals.org/ D ow nladed from demonstrated a 52 8% decrease (N 6, p 0.004) in expression of GIRK1 compared to WT. Adenoviral expression of SREBP1 in atrial myocytes from Akita mice restored the impaired carbamylcholine stimulated IKAch from -179 18.9 pA/pF (N 5) to -403 2 pA/pF (N 6, p 0.001) which was similar to that in cells from WT hearts. Taken together with further data demonstrating that the decreased response of the Akita mouse heart to parasympathetic stimulation is reversed by insulin, this study demonstrates a unique relationship between lipid homeostasis and the parasympathetic regulation of the heart via SREBP1 control of GIRK1 expression and IKAch activity which may offer a new therapeutic target for the treatment of this devastating complication of diabetes. Emerging Concepts in Cardiovascular Biology II: Late-Breaking Basic Science Poster Abstracts Arginase Regulates Uncoupling of the Nitric Oxide Synthase Dimer and Contibutes to Pulmonary Hypertension During Chronic Hypoxia in Mouse Lung Ryan J Tedford, Milena Gebska, Mobusher Mahmud, Hazim El-Haddad, Blake Stevenson, Hunter C Champion; John Hopkins Hosp, Baltimore, MD It has recently been reported that uncoupling of the functional NOS dimer plays a role in reducing NO production and increasing superoxide (O2) production, leading to dilatory heart failure under conditions of pressure overload. Here we establish that arginase, an enzyme that competes with NOS for the substrate L-arginine, contributes to the reduction in NOS dimerization and promotes pulmonary hypertension. Under conditions of chronic hypoxia, arginase protein abundance, mRNA expression, and enzyme activity were markedly elevated in mouse lung tissue (P 0.05), and the arginase activity was closely correlated to NOS coupling (dimerization and activity). Treatment with the arginase inhibitor, BEC, resulted in preservation of the NOS dimer and NOS activity and reduced expression of O2. In order to determine if the expression of arginase is causal in this loss of NOS coupling, adult mouse lungs were transfected with an AAV encoding arginase driven by either a Tie2 or -sm muscle actin promoter. Transfection led to a 4-fold increase in arginase activity when compared to lungs transfected with the reporter gene AAV gal. Ten days after transfection, there was a marked increase in O2 expression and a significant loss of NOS coupling that was prevented by treatment with BEC. Pulmonary arterial catheterization demonstrated significantly higher mean PA pressure at 6 weeks in AAV-arginase mice compared with AAV gal mice (19.8 1.1 mmHg compared to 13.9 0.9 mmHg, P 0.05). Arginase transfection was associated with a marked increase in ROS signaling, MMP activation, and pulmonary vascular remodeling. These results clarify the role of arginase in mediating pulmonary hypertension in vivo and provide the first evidence that arginase regulates NOS dimerization and activity. Pioglitazone Normalizes Function of Endothelial Progenitor Cells From Hypercholesterolemic Monkeys William B Strawn, Carlos M Ferrario; Wake Forest Univ Health Sciences, Winston Salem, NC Hypercholesterolemia promotes atherosclerosis by inducing endothelial dysfunction that may be augmented by the reduced reparative capacity of circulating endothelial progenitor cells (EPCs). Peroxisome proliferator-activated receptor (PPAR)agonists enhance hypercholesterolemiaassociated endothelial function by mechanisms that we hypothesized may include improved EPC function. To investigate this hypothesis, we determined whether the PPARagonist pioglitazone increases the functional activity of EPCs isolated from the circulation of normocholesterolemic (NC) and hypercholesterolemic (HC) monkeys. Adult male cynomolgus monkeys fed either a low cholesterol-containing diet (n 30) or a high cholesterol-containing diet (n 30) had total plasma cholesterol concentrations of 102 8 and 333 21 mg/dL, respectively. Primary cultures of EPCs were identified by morphology, uptake of acetylated LDL, and expression of endothelial lineagespecific markers. Function of EPCs in secondary culture was assessed by assays for cell proliferation, migration, vasculogenesis, senescence and apoptosis. The number of EPCs quantified by flow cytometry analysis was lower in the circulation of HC compared to NC monkeys (56.3 8.9 and 98.3 13.6 VEGFR2 /CD34 cells/100,000 events, respectively; P 0.05). Compared to NC monkeys, hypercholesterolemia decreased EPC proliferative and migratory capacities (by 64.6 6.3 and 59.2 5.1%, respectively; P 0.05), and vasculogenesis (by 42.4 6.7%; P 0.05). In contrast, EPC senescence and apoptosis were both increased in HC monkeys compared to NC monkeys (by 102 12.6 and 21.3 3.2%, respectively; P 0.05). Treatment of EPCs from NC monkeys with 10 M pioglitazone for 48 hours had no effect on any of the function parameters, whereas similar treatment of EPCs from HC monkeys normalized all function parameters. This study is the first to show that circulating EPC number and function are diminished compared to normocholesterolemic controls in a well-characterized nonhuman primate model of hypercholesterolemia-induced atherosclerosis. Furthermore, these results are the first to demonstrate that treatment with a PPARagonist reverses hypercholesterolemia-induced EPC dysfunction. Sex-Based Differences in Myocardial Calcium Regulation Rebecca E Petre, Michael P Quaile, TEMPLE Univ Sch OF MEDICINE, Philadelphia, PA; Sarah J Ratcliffe, Eric I Rossman, Univ OF PENNSYLVANIA Sch OF MEDICINE, Philadelphia, PA; Beth A Bailey, URSINUS COLLEGE, Philadelphia, PA; Steven R Houser, TEMPLE Univ Sch OF MEDICINE, Philadelphia, PA; Kenneth B Margulies; Univ OF PENNSYLVANIA Sch OF MEDICINE, Philadelphia, PA Recent studies support a central role for calcium-dependent signaling in pathological cardiac remodeling. Recognizing that there are sex differences in cardiac remodeling after pressure overload or myocardial infarction, we hypothesized that there are fundamental sex differences in calcium (Ca) regulation in cardiac myocytes. METHODS: Isometric force was measured at various frequencies (FFR) (0.5,1.0,1.5,2.0 Hz) during either changes in bath Ca ([Ca]o) (1.0,1.75,3.5,7.0 mM) or length (LT) (20,40,60,80,100% Lmax) in RV trabeculae from normal male (MT) and pre-menopausal female (FT) cats (n 37). RESULTS: Under basal conditions (0.5 Hz, 80% Lmax) both MT and FT achieved similar developed tension (MT 10.8 1, FT 10.2 1 mN/mm). However, at higher rates and preloads (2.0 Hz, 100% Lmax) MT exhibited greater force generation (MT 44.0 10.5, FT 27.8 5.6 mN/mm). Supplemental studies using rapid cooling contractures indicated greater sarcoplasmic reticulum Ca load in MT at higher frequencies (Fig 1). In contrast, force-Ca experiments in chemically skinned trabeculae indicated no sex difference in myofilament Ca sensitivity at a sarcomere length of 2.1 m ( 80% Lmax). CONCLUSION: Despite identical contractile performance under basal conditions, there are sex differences in myocardial contractile reserve during physiological stress. Our data demonstrate fundamental sex differences in cellular Ca regulation and SR Ca load that may affect Ca-dependent signaling and remodeling responses during sustained hemodynamic overload. Increased Apoptosis and Myocyte Enlargement in the Absence of Myocardial Hypertrophy: Distinctive Features of the Aged Male Monkey Heart David Lagunoff, Xiao-Ping Zhang, You-Tang Shen, Yimin Tian, UMDNJ New Jersey Med Sch, Newark, NJ; Ranillo R Resuello, Simian Conservation Breeding and Rsch Cntr, Manila, Philippines; Filipinas F Natividad, St. Lukes Med Cntr, Quezon City, Philippines; Stephen F Vatner, Dorothy E Vatner; UMDNJ New Jersey Med Sch, Newark, NJ Left ventricles were examined for myocyte cross-section area and frequency of apoptosis and Ki-67 expression in young and old Macaca fascicularis monkeys of both sexes. Mean myocyte cross-section area increased 54% in old males. Surprisingly, in contrast to load-induced left ventricular hypertrophy, the average weight of old male left ventricles showed a nonsignificant decrease from that in young adult males. A second major finding of the myocyte hypertrophy of aging was the character of the distribution of myocyte size. The increase in mean cross-section area was associated with positive skewing and kurtosis and a significant increase in the coefficient of variation of the distribution, not known to occur in load-induced left ventricular hypertrophy. In contrast, the average weight of the old female left ventricles exhibited a small increase associated with a comparable modest increase of 17% in mean myocyte cross-section area. The change in myocyte cross-section area in old males accompanied a 7 fold increase in frequency of apoptosis. There was no increase in apoptosis in the old female monkey hearts. The frequency of proliferation-capable myocytes, as assessed by Ki-67 expression, was greater in old male than old female monkeys, but not greater than in young males and clearly not capable of maintaining left ventricle mass. These results indicate a distinct difference in the programs in male and female monkeys for the maintenance of functional heart mass during aging with the old male hearts exhibiting increased apoptosis and myocyte enlargement in the absence of cardiomegaly, and the females relying on reduced apoptosis. Ottawa Heart Genomics Study: The First Genome-Wide Scan to Search for CAD Genes Utilizing 500,000 Markers Alexandre F Stewart, Ruth McPherson, Kathryn Williams, George Wells, Julie Rutberg, Heather Doelle, Gwen Ewart, Yanqing Wang, Lan Vo, Sybill Hebert, Thet Naing, Nihan Kavaslar, Marino Labinaz, Miguel Andrade, Robert Roberts; UNIV OF OTTAWA HEART INSTITUTE, Ottawa, Canada Background: Coronary artery disease (CAD) remains the number one killer in the western world. Genetics account for over 50% of the risk for CAD. Genetic screening and early prevention in individuals identified at increased risk could dramatically reduce CAD, thus, the necessity to identify genes predisposing to CAD. Genes identified by the candidate gene approach have not been replicated due in part to inadequate sample size. Genome wide scan association studies have been limited by the use of thousands of markers rather than the hundreds of thousands required and hundreds of individuals rather than the thousands required (Science 2005;307:1072). Replication of positive findings in an independent population is essential. To detect a Minor Allele Frequency of 5%, an odds ratio for risk of 1.3, with 90% power, we estimate 14,000 (9,000 affected and 5,000 control) subjects are required (Nat Rev Genet, 2005;6:95). Methods: The Affymetrix 500,000 marker set provides a marker every 6,000 base pairs as required and is being used to genotype 1,000 cases of premature CAD and 1,000 normals followed by replication in 8,000 cases and 4,000 controls. The phenotype is confirmed or excluded by coronary arteriograms by catheterization or multi-slice CT. Results: 1280 Circulation Research Vol 99, No 11 November 24, 2006 Basic Science by gest on Sptem er 4, 2017 http://circhajournals.org/ D ow nladed from Initiated in 2005, over 542 million genotypes have been analyzed for 595 controls and 505 cases. On average each DNA sample analyzed provided accurate and interpretable genotypes for 98.7% of the 500,000 SNPs. Cases and controls in Hardy-Weinberg equilibrium exhibited over 9,176 SNPs with a difference in allele frequency (P 0.001). We have identified 17 non-synonymous cDNA polymorphisms, 237 gene-specific clusters and 11 intergenic clusters associated with CAD. Genes containing SNPs or near a SNP associated with CAD are being assessed for their function in vitro (cell culture) and in vivo (transgenic animals). Conclusions: This is the first genome wide scan for CAD genes utilizing a marker every 6,000 base pairs. Several genes associated with CAD have been identified. LRP1 Controls the Activation State of Vascular Smooth Muscle Cells During Atherogenesis Through PDGFR -Dependent Regulation of Phosphoinositide-3 Kinase Li Zhou, Yoshiharu Takayama, Michelle Tallquist, Joachim Herz; UT Southwestern, Dallas, TX Low density lipoprotein receptor-related protein 1 (LRP1) protects against atherosclerosis by controlling the expression and activation of platelet-derived growth factor receptor (PDGFR ) in vascular smooth muscle cells (SMC). Activated PDGFR interacts with a wide range of signaling molecules, among which phosphoinositide-3 kinase (PI3K) is instrumental for initiating DNA synthesis and cell migration. In the present study, we show that SMC-specific LRP1 deficiency (smLRP1-/-) in mice shifts the SMC from the “contractile” to the “synthetic” state, resulting in SMC hyperproliferation and thickening of the aortic wall. Increased expression and tyrosine phosphorylation of PDGFR results in increased binding of PI3K to the receptor and leads to pronounced atherosclerotic lesion formation in the absence of hypercholesteremia. To investigate whether LRP1 affects atherogenesis by regulating the activation status of SMCs specifically through PDGFR -dependent PI3K activation, we generated a strain of smLRP1-/mice in which tyrosines 739 and 750 of the PDGFR had been mutated to phenylalanines (PDGFR F2/F2). Atherosclerosis is significantly reduced in these mice compared to smLRP-/mice that express wild type PDGFR . In vitro analysis of cultured primary SMCs shows significantly accelerated proliferation and migration when LRP is absent. By contrast, SMCs from smLRP-/mice expressing the mutant PDGFR F2/F2 show dramatically reduced cell proliferation and migration. Taken together, our findings show that LRP1 regulates SMC proliferation and migration by controlling PDGFR -dependent activation of PI3K and this is essential for preventing atherosclerosis and maintaining vascular integrity. Postnatal Cardiomyocyte-Restricted Knockout of a COP9 Signalosome Gene Compromises Proteolytic Function of the Ubiquitin Proteasome System and Causes Congestive Heart Failure in Mice Huabo Su, Univ South Dakota, Vermillion, SD; Suchithra Menon, Yale Univ, New Haven, CT; Kathleen M Horak, Jie Li, Univ South Dakota, Vermillion, SD; Faqian Li, South Dakota Health Rsch Foundation, Sioux Falls, SD; Ning Wei, Yale Univ, New Haven, CT; Xuejun ( Wang; Univ South Dakota, Vermillion, SD The ubiquitin-proteasome system (UPS) degrades all abnormal proteins and most unneeded normal proteins in the cell. It is extremely important in both protein quality control and homeostasis of the cell. UPS proteolytic function in the heart can be impaired by abnormal protein aggregation which is prevalent in failing human hearts. Thus, it is suggested that UPS dysfunction is very important in the pathogenesis of congestive heart failure (CHF) and may represent a new potential therapeutic target. To target cardiac UPS, we must understand how the UPS is regulated in the heart. The COP9 signalosome (CSN), a recently discovered protein complex consisting of 8 unique protein subunits (CSN1 CSN8), is likely a key regulator of the UPS. All germ-line knockouts of CSN genes are early embryonic lethal, preventing the CSN from being studied in the heart or any organs. Here we report that we have recently achieved postnatal cardiomyocyte-restricted knockout of the Csn8 gene (CR-Csn8KO) in mice using the cre-lox system. CR-Csn8KO occurred between postnatal day 2 and day 7 in mice with a genotype of Csn8 coupled with transgenic Cre driven by the alpha-myosin heavy chain promoter. CR-Csn8KO mice started to show increased heart/body weight ratios at 2 wks, increased lung/body weight ratios and body growth arrest after 3 wks of age. They died of CHF at 1 month of age. Echocardiography showed left ventricle wall thinning and chamber dilatation and decreases in the ejection fraction at 3 wks. Caspase activation was evident in Csn8KO cardiomyocytes. Electron microscopy of myocardium revealed marked defects in myofibrilogenesis. CR-Csn8KO caused significant decreases in both the CSN holo-complexes and the protein levels of other CSN subunits. De-neddylation of cullins is critical to the dynamics of the cullin-based E3 ligases, the largest family of ubiquitin ligases. CR-Csn8KO accumulated neddylated cullin 1 and 4. Crossing with UPS reporter (GFPdgn) mice reveals that UPS function is significantly impaired in CR-Csn8KO hearts before cardiac failure is discernible. Our data support the hypothesis that Csn8 is essential to CSN complex formation and UPS function in the heart and CSN8 is required for postnatal cardiac development, myofibrilogenesis, and cardiac function. Gene Therapy With PKG1Reduces Myocardial Infarction Following Ischemia/Reperfusion in Mice Fadi N Salloum, Anindita Das, Evan D Ownby, Rakesh C Kukreja; VIRGINIA COMMONWEALTH UNIV, Richmond, VA Background: Cyclic GMP-dependent protein kinases (PKGs) are key mediators of cGMP signaling in the heart. Ischemic and pharmacologic preconditioning against ischemia/ reperfusion injury (I/R) have been shown to involve upstream regulating elements controlling NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection. However, the direct effect of PKG in cardioprotection without the confounding factors associated with preconditioning stimuli or drugs is not known. We therefore studied the effect of PKG1gene therapy on myocardial infarction following I/R. Methods and Results: Adult ICR mice (28–33g) were injected with adenoviral vector encoding PKG1(10 pfu/mouse) or vehicle in the left ventricular wall 96 hr prior to heart explantation for molecular analyses or regional ischemia by left coronary artery ligation for 30 min and reperfusion for 24 hr. At the end of reperfusion, infarct size (IS) was measured using computer morphometry of tetrazolium chloride stained sections. Myocardial IS (mean SE) was significantly reduced in mice over-expressing PKG as compared with control group (Fig. 1A; 61% decline). The risk area was not different between groups (Fig. 1B). Real time PCR data showed a significant increase in PKG1message in the over-expressed group versus control (Fig. 1C) which was confirmed by WB analysis with profound up-regulation of PKG1protein (Fig. 1D). Conclusion: This study provides first evidence that direct over-expression of PKG1in vivo reduces myocardial infarction following I/R. We propose that up-regulation of PKG may be a novel therapeutic strategy to suppress I/R injury in patients with cardiovascular disease. ABCG1 Protects Against Atherosclerotic Lesion Development Ruud Out, Menno Hoekstra, Illiana Meurs, Paula de Vos, Johan Kuiper, Miranda Van Eck, Theo J J Van Berkel; UNIV OF LEIDEN, Leiden, The Netherlands In bone marrow transplantation studies, we and others have very recently shown that the absence of macrophage ABCG1 may differentially influence atherosclerosis. To further define the role of ABCG1 in atherogenesis, we investigated the effect of total body deficiency of ABCG1 on atherosclerotic lesion development by feeding ABCG1 mice and ABCG1 / mice an atherogenic diet for 12 weeks. Before and after the diet, serum lipid levels and lipoprotein profiles did not differ significantly between the two groups. In ABCG1 / mice the diet induced the formation of macrophage-rich early lesions (size: 24 7x10 m (n 6)). Feeding ABCG1 mice the atherogenic diet led to a significant 1.9-fold stimulation of atherosclerotic lesion size (46 6x10 m (n 7); p 0.034) compared to ABCG1 / mice, suggesting a clear antiatherogenic role for ABCG1. At the same time, excessive lipid accumulation was observed in macrophage-rich areas of the lungs and spleens of ABCG1 mice. When the fold increase in atherosclerotic lesion size of ABCG1 mice as compared to ABCG1 / mice is plotted against total serum cholesterol values, including recently reported data, a highly significant inverse correlation is found (R 0.95, P 0.005), whereby at about 900 mg/dL serum cholesterol a switch from ABCG1’s protective function to lesion promotion is noticed, probably due to compensatory mechanisms (see figure). In conclusion, both macrophage-specific and total body ABCG1 deficiency studies show increases in lesion development at serum cholesterol values relevant for the human situation, which points towards an atheroprotective effect of ABCG1 in human atherosclerotic lesion formation. Simultaneous Administration of Darbepoetin Alpha and Insulin Growth Factor-1 Protects the Rat Myocardium Against Myocardial Infarction and Enhances Angiogenesis Erhe Gao, Matthieu Boucher, Stephanie Pesant, J Kurt Chuprun, Rui-Hai Zhou, Andrea D Eckhart, Walter J Koch; Thomas Jefferson Univ, Philadelphia, PA BACKGROUND: Recent studies showed that insulin growth factor-1 (IGF-1) and either erythropoietin (EPO) or the long-acting EPO analogue Darbepoetin alpha (DA) protects the heart Basic Science Late Breaking Abstracts 1281 by gest on Sptem er 4, 2017 http://circhajournals.org/ D ow nladed from against ischemia/reperfusion (I/R) and myocardial infarction (MI). The present study examined the cardioprotective effect of simultaneous treatments with IGF-1 and DA in these models. METHODS: Male adult rats were subjected to 30 minutes ischemia and 72 hours reperfusion (I/R) or 4 weeks of ischemia alone (MI). Four different groups were constituted for each model: Vehicle, IGF-1 (1 g/kg), DA (30 g/kg for the I/R model or 20 g/kg for the MI model), and IGF-1 DA. Treatments were administered just before the onset of ischemia. For the MI model, IGF-1 (1 g/kg) and/or DA (10 g/kg) was administered weekly. At the appropriate time point cardiac function (hemodynamics and echocardiography) and myocardial infarction were measured. Apoptosis was quantified via TUNEL staining and caspase-3 activity. Angiogenesis was determined via Von Willebrand factor immunohistochemistry staining. RESULTS: IGF-1 or DA treatments alone reduced significantly infarct size following both I/R and MI. Simultaneous treatments had a similar protective effect on infarct size. Treatments led to improvement of cardiac function after I/R or MI (enhanced dP/dt and ejection fraction) compared to Vehicle. After I/R, apoptosis was reduced with either or both IGF-1 and DA treatments, as measured by reduced TUNEL staining and caspase-3 activity. In addition, after 4 weeks of ischemia alone (MI), DA induced angiogenesis. Furthermore, simultaneous treatments with IGF-1 and DA enhanced angiogenesis significantly compared to vehicle or either treatments alone. CONCLUSION: These data indicate simultaneous pharmacological treatment with IGF-1 and DA protects the heart against I/R and MI injuries. This protection results in reduced infarct size and improved cardiac function. Moreover, this treatment reduces apoptosis and enhances angiogenesis in the ischemic heart. Novel Inhibitor of N-Ethylmaleimide-Sensitive Factor Protects Against Myocardial Ischemia-Reperfusion Injury John W Calvert, Albert Einstein College Of Medicine, Bronx, NY; William M Baldwin, III, The Johns Hopkins Univ Sch of Medicine, Baltimore, MD; David J Lefer, Albert Einstein College Of Medicine, Bronx, NY; Charles J Lowenstein; The Johns Hopkins Univ Sch of Medicine, Baltimore, MD Background: Exocytosis of endothelial granules (i.e., Weibel-Palade bodies) may contribute to reperfusion (R) injury following myocardial ischemia (MI). Weibel-Palade bodies contain von Willebrand’s factor (vWF) and P-selectin. These proteins promote the activation, aggregation, and adhesion of both platelets and leukocytes. Several proteins mediate exocytosis, however, N-ethylmaleimide-sensitive factor (NSF) plays a critical role in regulating this process. TAT-NSF700 is a novel peptide that reduces endothelial exocytosis by inhibiting the ATPase activity and disassembly activity of NSF. We hypothesized that TAT-NSF700 would limit myocardial injury in an in vivo murine model of MI-R. Methods: Mice (n 12/group) were subjected to 30 min of MI followed by 24 hrs of R. TAT-NSF700 (0.5 mg/kg) or the scrambled peptide, TAT-NSF700scr (0.5 mg/kg), was administered (i.v.) 20 min prior to the onset of ischemia. At 24 hrs of reperfusion, hearts were excised and evaluated for infarct (INF) size. Endothelial exocytosis was evaluated by the extent of vWF staining in the injured myocardium 20 min following reperfusion. Results: TAT-NSF700 treatment attenuated myocardial infarct size by 45% with respect to the left ventricle (INF/LV) and by 47% relative to the area-at-risk (INF/AAR). Furthermore, TAT-NSF700 significantly (P 0.05) decreased the release of vWF in the injured myocardium in the early phase of reperfusion. Conclusions: These data suggest that inhibiting endothelial exocytosis may prove beneficial in attenuating the extent of myocardial necrosis following MI-R. The Role of O-GlcNAcylation in Aging-Related Loss of Estrogenic Vasoprotection Andrew P Miller, Cheryl Robertson, Dongqi Xing, Peng Li, Yiu-Fai Chen, John C Chatham, Suzanne Oparil; Univ Alabama Birmingham, Birmingham, AL Background: Posttranslational modification of nucleocytoplasmic proteins by addition of O-linked -N-acetylglucosamine (O-GlcNAc) to Ser and Thr residues by the enzyme O-GlcNAc transferase (OGT) has been shown to modulate a variety of cellular functions, including responses to stress/trauma. We have previously demonstrated that 17 -estradiol (E2) has anti-inflammatory and vasoprotective effects in injured arteries of young ovariectomized (OVX) female rats that are lost with aging. In the current study, we tested the hypotheses that (1) E2 treatment stimulates protein O-GlcNAcylation in vascular tissues of young OVX rats, and that (2) this response is lost in aged animals. Methods: Young (10 wk-old) and aged (22 mo-old) female Sprague-Dawley rats were studied intact or 5 days after OVX and treatment with either E2 or vehicle (V). Rats were sacrificed within 4 hrs of the last injection, serum collected for assessment of E2, glucose, insulin and lipid levels, and tissues harvested for quantification of tissue OGT and O-GlcNAc protein expression by Western Blot analysis. Results: O-GlcNAc protein levels were markedly increased in carotid arteries (2.75-fold, p 0.0001) and hearts (2-fold, p 0.002) from intact aged compared with young animals, without concomitant alterations in OGT expression. E2 treatment significantly increased O-GlcNAc protein expression in carotid arteries (by 27%, p 0.04) of young but not aged animals. Conclusion: There is an aging-related accumulation of O-GlcNAcylated proteins in cardiovascular tissues that is not accounted for by changes in OGT expression. E2 induces acute increases in O-GlcNAc modified proteins in carotid arteries from young but not aged animals. O-GlcNAcylation may play an important role in the acute estrogenic stress-modulating responses seen in carotid arteries from young but not aged subjects.