HomeCirculation ResearchVol. 111, No. 122012 Late-Breaking Basic Science Oral Session Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUB2012 Late-Breaking Basic Science Oral Session Originally published2 Nov 2012https://doi.org/10.1161/RES.0b013e318274d3e1Circulation Research. 2012;111:e379–e387Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2012: Previous Version 1 Late-Breaking Basic Science Oral Session22925Cardiac Myosin Binding Protein C is an Ultra-early and Cardiac-specific Biomarker of Myocardial NecrosisDiederik W Kuster1, Adriana Cardenas-Ospina2, Lawson Miller2, Christian Troidl3, Holger M Nef3, Christoph Liebetrau3, Möllmann Helge3, Karen S Pieper4, Kenneth W Mahaffey4, Neal S Kleiman5, Bruno D Stuyvers2, Ali J Marian6, Sakthivel Sadayappan1; 1Loyola Univ Chicago, Maywood, IL, 2Memorial Univ, St John’s, Canada, 3Kerckhoff Heart and Thorax Cntr, Bad Neuheim, Germany, 4Duke Univ Med Cntr, Durham, NC, 5The Methodist DeBakey Heart and Vascular Cntr, Houston, TX, 6Univ of Texas Health Sciences, Houston, TXRationale: We recently demonstrated that proteolytic cleavage fragments of cardiac myosin binding protein-C (cMyBP-C) can be detected in the serum after myocardial infarction (MI) in a rat model and in patients with MI. The findings implied that serum cMyBP-C might be useful as cardiac-specific biomarker for MI. The release kinetics of cMyBP-C in the circulation post-MI remains to be elucidated. Objective: Determine the release kinetics of cMyBP-C as an ultra-early and cardiac-specific biomarker of myocardial necrosis. Method and Results: To determine the exact timing of cMyBP-C release in the bloodstream post-acute MI, left anterior descending (LAD) coronary artery was ligated in adult swine (n=6). ECG showed significant ST elevation. Infarct size represented 12.4 ± 1.9% of total ventricular mass. Blood samples were collected before and at predetermined time points between 30 min and 14 days after LAD ligation. Plasma cMyBP-C level was quantified using a highly sensitive and rapid sandwich enzyme-linked immunosorbent assay. Compared with baseline, cMyBP-C levels were increased in post-MI serum within 45 min (0.64 ± 0.52 ng/ml) after LAD ligation and declined after 16 hrs to the baseline level (0.01 ± 0.00 ng/ml). In contrast, cardiac troponin I (cTnI) level peaked after 6 hrs and returned to baseline after 10 days. To validate these findings in humans, serial blood samples were taken from 5 patients with hypertrophic cardiomyopathy undergoing transcoronary ablation of septal hypertrophy (TASH). Similar to the swine model, the level of cMyBP-C increased 30 min after TASH (0.25 ± 0.15 ng/ml) and peaked at 4 hrs (0.56 ± 0.27 ng/ml), confirming that cMyBP-C is a promising ultra-early biomarker of MI. Furthermore, cMyBP-C level was determined in patients with acute coronary syndromes (ACS) from the SYNERGY library population and a healthy control group (n=160 and 61, respectively). Seventy-eight percent (125 out of 160) of patients with ACS had detectable cMyBP-C serum levels (2.9 ± 1.2 ng/ml), implicating serum cMyBP-C as a biomarker for ACS. Conclusion: The rapid appearance of proteolyzed cMyBP-C in the circulation post-acute MI in a swine model and in human patients with ACS and post-TASH identify serum cMyBP-C as an ultra-early biomarker of myocardial necrosis.Author Disclosures: D.W.D. Kuster: None. A. Cardenas-Ospina: None. L. Miller: None. C. Troidl: None. H.M. Nef: None. C. Liebetrau: None. M. Helge: None. K.S. Pieper: None. K.W. Mahaffey: None. N.S. Kleiman: None. B.D. Stuyvers: None. A.J. Marian: None. S. Sadayappan: None.Key Words: Myocardial infarction; Biomarkers; Myocardial infarction, NSTEMI; Acute coronary syndromes; Contractile proteins22626Replacement Of Mybpc3 Mutation By 5’-trans-splicing In A Knock-in Mouse Model: A Step Towards Causal Therapy Of Hypertrophic CardiomyopathyDoreen Khajetoorians1, Giulia Mearini1, Elisabeth Kraemer1, Birgit Geertz1, Christina Hornung1, Oliver Mueller2, Thomas Eschenhagen1, Thomas Voit3, Luis Garcia3, Stephanie Lorain3, Lucie Carrier1; 1Univ Med Cntr Hamburg-Eppendorf, Hamburg, Germany, 2Univ Hosp Heidelberg, Heidelberg, Germany, 3Inserm U974, Paris, FrancePurpose: Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric septal hypertrophy, diastolic dysfunction, myocardial disarray and lacks curative treatment. It is often caused by mutations in MYBPC3 encoding cardiac myosin-binding protein C (cMyBP-C). Most of the mutations alter mRNA splicing and result in aberrant mRNAs and proteins. In the present study we evaluated the feasibility and efficacy of RNA correction using spliceosome-mediated 5’-trans-splicing to remove the mutation in vitro and in vivo in Mybpc3-targeted knock-in (KI) mice. Methods and Results: KI mice carry a G > A transition in exon 6, which results in low levels of mutant Mybpc3 mRNAs and cMyBP-C proteins. We generated a series of FLAG-tagged pre-trans-splicing molecules (PTM) containing wild-type exons 1-6 with binding domains complementary to intron 6 of Mybpc3. The PTMs were packaged in adeno-associated virus serotype 6 or 9 (AAV6; AAV9) driven by a cardiomyocyte-specific promoter. Cardiac myocytes isolated from neonatal KI mice (NMCMs) were transduced with AAV6 for 7 days and AAV9 was systemically administered in 3-day-old KI mouse for 7 weeks. The efficacy of 5’-trans-splicing was evaluated by RT-PCR, Western Blot and immunofluorescence. The trans-spliced mRNA was amplified with specific primers only in PTM-transduced samples in vitro and in vivo. The correction of the mutation was confirmed by sequencing. By semi-quantitative PCR trans-spliced mRNA was estimated to represent 33% and 0.14% of total Mybpc3 transcripts in vitro and in vivo, respectively. Whereas the trans-spliced cMyBP-C protein was not revealed by Western blot, up to 9% of FLAG-positive striated cardiac myocytes were detected by immunofluoresence and exhibited correct incorporation of trans-spliced cMyBP-C in doublets in the A-band of the sarcomere. Conclusion: These data provide the first proof-of-concept of 5’-trans-splicing to correct cardiac genetic defects in vitro as well as in vivo. Therefore, spliceosome-mediated 5’-trans-splicing may be a promising approach for the treatment of HCM.Author Disclosures: D. Khajetoorians: None. G. Mearini: None. E. Kraemer: None. B. Geertz: None. C. Hornung: None. O. Mueller: None. T. Eschenhagen: None. T. Voit: None. L. Garcia: None. S. Lorain: None. L. Carrier: None.Key Words: Hypertrophic cardiomyopathy; Gene therapy22911Hematopoietic Id Ablation Contributes to Pathogenesis in the Adult HeartCorey Chang, Qingshi Zhao, Diego Fraidenraich; Univ of Medicine and Dentistry of New Jersey, Newark, NJThe Id (inhibitor of DNA binding) genes play a crucial role in cardiovascular development. Id double knockout (Id dKO) embryos lacking both Id1 and Id3 develop multiple cardiac defects reminiscent of the “thin myocardial wall” syndrome and are lethal at mid-gestation. While Id genes are only expressed in non-myocardial layers (epicardium, endocardium, endothelium), the myocardium is affected suggesting a paracrine mechanism of action. Maternal injection of IGF1 (epicardial Id-dependent signal) failed to rescue inner heart defects seen in Id dKO embryos, suggesting that the endocardium/endothelium play an important role in cardiac development. To circumvent embryonic lethality and study the role of Id in the endocardium/endothelium in adult mice, we generated a conditional knockout (Id cKO) using the Tie2Cre/loxP system. These mice are Id3 null with endocardial/endothelial-specific Id1 ablation. Surviving Id cKOs develop dilated fibrotic cardiomyopathy, hematopoietic defects and splenomegaly in adulthood, suggesting that Id loss in Tie2 expressing organs may be responsible. Since Tie2 is also expressed in hematopoietic cells, it is unclear if loss of hematopoietic Id contributes to cardiac pathology in Id cKOs. To address this question, we transplanted WT GFP-labeled bone marrow into lethally irradiated Id cKOs (WT/Id cKOs) to test if a normal hematopoietic system can ameliorate aspects of cardiomyopathy in our model (rescue). We also performed the reverse experiment (Id cKO/WTs) to test if an Id-deficient hematopoietic system negatively impacts a normal heart (dysregulation). Full bone marrow reconstitution occurred. Within the endomyocardium, we found marked acellularity and fibrosis in Id cKOs, decreased fibrosis and improved cellularity in WT/Id cKOs, and emerging interstitial fibrosis and mononuclear invasion in Id cKO/WTs. The marked perivascular fibrosis in Id cKO hearts was reduced in WT/Id cKO hearts. Ejection fraction decreased in Id cKOs, improved in WT/Id cKOs and decreased in Id cKO/WTs (64.4+/-5.7% n=5 WT/Id cKOs; 58.4+/-7.97% n=9 Id cKO/WTs; 56.2+/-13.2% n=56 Id cKOs; 64.3+/-6.8% n=15 WTs). These results suggest that Id levels in bone marrow cells play an important role in the development of dilated, fibrotic cardiomyopathy.This research has received full or partial funding support from the American Heart Association.Author Disclosures: C. Chang: None. Q. Zhao: None. D. Fraidenraich: None.Key Words: Cardiovascular development; Adult congenital heart disease; Vascular disease-Cardiomyopathy; Heart development22955Detection And Therapy Of Ischemia-reperfusion Injury Using Hydrogen Peroxide-responsive Molecularly Engineered Polymer NanoparticlesDongwon Lee1, Soochan Bae2, Donghyun Hong3, On Hwang2, Seunggyu Park2, Joo H Yoon2, Qingen Ke2, Peter M Kang2; 1Chunbuk National Unversity, Jeonju, Korea, Republic of, 2BETH ISRAEL DEACONESS MED CTR, Boston, MA, 3Chunbuk National Univ, Jeonju, Korea, Republic ofThe main culprit in the pathogenesis of ischemia/reperfusion (I/R) injury is the overproduction of hydrogen peroxide (H2O2), a causal agent for apoptosis and inflammation that lead to cellular damage and organ dysfunction. In this study, we generated and characterized novel H2O2-responsive nanoparticles formulated from polyoxalate co-polymer containing vanillyl alcohol (PVAX) during I/R injury. PVAX, in the presence of H2O2, degrades completely into three safe compounds, cyclohexanedimethanol, VA and CO2. PVAX effectively scavenges H2O2 in a dose-dependent manner, with the intrinsic anti-oxidant and anti-inflammatory properties in vitro. Using In Vivo Imaging System, we demonstrated that PVAX loaded with rubrene as a fluorophore robustly imaged H2O2 generated during I/R in the hind-limb I/R injury in vivo, demonstrating their potential for bioimaging of H2O2. PVAX nanoparticles injected into the site of hind-limb I/R injury also exerted highly potent anti-inflammatory and anti-apoptotic activities resulting in a significantly less cellular damage compared to the vehicle-treated group. We then examined the effect of PVAX in doxorubicin (DOX)-mediated cardiomyopathy since a major mechanism of DOX-mediated toxicity has been shown to involve increased oxidative stress. We found that i.p. injection of 100 µg of PVAX daily resulted in significant decrease in apoptosis as well as PARP-1 and caspase activation in both heart and liver compared to vehicle treat ed groups. Furthermore, 10 days after DOX administration PVAX treated animals, compared to vehicle treated animals, demonstrated 31% less decrease in body weight, 28% improvement in cardiac function and significantly improved survival rate (Veh= 35% vs PAVX=71%; p<0.05%). Moreover, PVAX administered daily for 7 days demonstrated no histological or functional abnormalities in various organs. Thus, we conclude that PVAX is a novel multifunctional nanoparticle that possesses intrinsic anti-oxidant and anti-inflammatory properties, and has a tremendous potential to be used as a theranostic agent for I/R injury in heart and other organs.Author Disclosures: D. Lee: None. S. Bae: None. D. Hong: None. O. Hwang: None. S. Park: None. J.H. Yoon: None. Q. Ke: None. P.M. Kang: Research Grant; Significant; Abbott Laboratories.Key Words: Ischemia reperfusion; Apoptosis Inflammation; New technology; Cardiomyopathy22594Communication with Cardiomyocytes in Contact Co-culture Reprograms Mesenchymal Stem Cells for Improved CardiomyogenesisVien Khach Lai, Shujia Jiang, Kelly S Prindle, Khawaja H Haider; Univ of Cincinnati, Cincinnati, OHBackground: Bone marrow mesenchymal stem cells (MSC) can differentiate to morpho-functional cardiomyocytes (CM) when maintained in conductive culture conditions in vitro and post-transplantation in the ischemic heart albeit with an as yet undefined mechanism. We hypothesized that propensity of MSC for reprogramming to adopt cardiac phenotype and their myocardial reparability could be enhanced if the cells were co-cultured with CM in vitro prior to transplantation. Methods and results: Bone marrow cells were isolated from GFP expressing transgenic adult rats and analyzed for MSC specific surface markers by flow cytometry. The cells were co-cultured with PKH-26 labeled rat neonatal CM at 1:3 ratios in a dual chamber dish separated by a semi-permeable membrane or by direct contact co-culture for 0-4 days. Conventional fluorescence microscopy revealed formation of nano-tubular structures between MSC in contact co-culture with CM. Live cell imaging showed transient exchange of cytosolic contents between the two juxtaposed cell types which was indicated by PKH26 red fluorescence dye transfer. Flow cytometry revealed increasing percentage of double positive GFP+ MSC in time-dependent manner (up to 81.08 ± 5.91% of GFP+ MSC received the red dye transferred from CM at day 3 of co-culture). Cardiac specific gene expression including GATA4, Nkx2.5 and MEF2c was elevated in MSC at 3 days after co-culture with CM. These results were substantiated by immunocytochemistry. Transplantation of MSC (co-cultured for 3 days) in a rat heart model of acute coronary artery ligation resulted in significantly reduced infarction sizes (38.2 ± 3.1 vs 25.0 ± 3.8 for control vs co-cultured MSC, p<0.05) and preserved global heart function (left ventricular ejection fraction: 46.0 ± 1.2 vs 56.1 ± 4.0% for control vs co-cultured MSC, p<0.05) after 3 weeks of MSC transplantation compared to that of control. Conclusion: Direct contact with CM (1:3) in co-culture reprograms MSC for enhanced rate of cardiomyogenic differentiation via exchange of their cytosolic contents. These findings provide an insight into the mechanisms which underlie the morpho-functional cardiogenic differentiation of MSC post-transplantation in an infarcted heart.Author Disclosures: V. Lai: None. S. Jiang: None. K.S. Prindle: None. K.H. Haider: None.Key Words: Myocardial infarction; Regenerative medicine stem cells; Transplantation; Molecular biology22894Telomeric Shortening Induces a Senescent Cardiomyopathy that is Independent from Chronological ageJunghyun Kim, Fumihiro Sanada, Maria Cimini, Christian Arranto, Polina Goihberg, Toru Hosoda, Marcello Rota, Jan Kajstura, Piero Anversa, Annarosa Leri; Brigham and Women’s Hosp, Boston, MATelomeric shortening is viewed as the hallmark of organism, organ, and cellular senescence. It remains, however, to be documented whether loss of telomeric DNA in cardiac stem cells (CSCs) and/or cardiomyocytes is causally involved in the manifestations of the aging cardiomyopathy. To address this issue, the heart of mice carrying a deletion of the RNA component of telomerase (Terc−/− mice) was studied at 3-7 months of age. This allowed us to define whether telomere shortening in stem cells and their progeny promotes ventricular dysfunction independently from chronological age. The structural and functional characteristics of the aging cardiomyopathy were defined initially in senescent 30-month-old wild-type mice (WT). By echo-Doppler, MRI, and invasive hemodynamics, deterioration in systolic and diastolic indices of myocardial contractility were detected in these senescent mice. Quantitative parameters obtained by MRI documented the presence of chamber dilation and attenuated ventricular torsion capacity and diastolic strain rate. With respect to young mice, a 50% reduction in telomere length was detected in CSCs and cardiomyocytes isolated from 30 month-old mice. Importantly, 7-month-old Terc−/− mice showed severe ventricular dysfunction comparable to that seen in 30 month-old WT. Telomere length in Terc−/− mouse CSCs and myocytes was ~50-70% shorter than in age-matched WT cells but was comparable to that detected in 30 month-old WT cells. The number of CSCs was 60% lower in Terc−/− than age-matched WT mice, and the fraction of BrdU-positive CSCs decreased 1.4-fold, from 25% to 14%. The absence of Terc led to a 50% reduction in myocyte turnover, which was coupled with myocyte hypertrophy and significant myocyte loss. BrdU and Ki67 labeling were reduced, respectively, by 65% and 60% in Terc-/- myocytes. Old CSCs formed a senescent progeny composed of cardiomyocytes, which carried markedly shortened telomeres, were consistently larger in volume, and showed a severe depression in cell shortening and re-lengthening. Our findings document that telomeric shortening is the critical determinant of CSC aging and reduced myocyte renewal. The accumulation of senescent CSCs and cardiomyocytes leads to pathological cardiac remodeling and heart failure.Author Disclosures: J. Kim: None. F. Sanada: None. M. Cimini: None. C. Arranto: None. P. Goihberg: None. T. Hosoda: None. M. Rota: None. J. Kajstura: None. P. Anversa: None. A. Leri: None.Key Words: Stem cells; Aging; Cardiac regeneration22950Identification of Novel Non-steroidal Modulators of Vitamin D Receptor with Cardioprotective Property without Hypercalcemic EffectJi Yoo Lee1, Santosh A Khedkar1, Sangita Choudhury1, Mohammed Samad1, Dongsheng Zhang1, Ravi I Thadhani2, S A Karumanchi1, Alan C Rigby1, Peter M Kang1; 1BETH ISRAEL DEACONESS MED CTR, Boston, MA, 2Massachusetts General Hosp, Boston, MAVitamin D is a multifunctional, steroid hormone responsible for regulating various biological processes. Vitamin D therapy has been shown to prevent cardiac hypertrophy and improve cardiac dysfunction. However, its clinical utility has been limited by hypercalcemia. In this study, we report on a novel vitamin D agonist tool compound discovered using computer aided drug discovery (CADD) that possesses significant anti-hypertrophic activity without hypercalcemia. Using this CADD approach, we screened approximately 4 million unique compounds virtually using chemical features/descriptors from known vitamin D receptor (VDR) agonists that had minimal demonstrable hypercalcemia activity. These hit compounds were vetted using an ensemble structure-based screen of the VDR. The top 174 CADD molecules were evaluated using the GeneBLAzer® Cell-Based VDR Assay to identify those compounds that significantly regulated the transcriptional profile of VDR; we identified 5 compounds that showed considerable activation at 100 µM or lower. The compound possessing the greatest VDR binding activity (known as VDR136) showed a significant concentration-dependent suppression of phenylephrine (PE)-induced cardiac hypertrophy in adult cardiomyocyte culture in vitro and mouse infused with PE via osmotic pump in vivo. In addition, VDR136 significantly suppressed cardiac hypertrophy and progression to heart failure induced by transverse aortic constriction (TAC) as compared to the vehicle treated group as determined by 17% decrease in HW/BW ratio and 41% improvement in fractional shortening. VDR 136 also demonstrated effective suppression secondary hyperparathyroidism in 1α-hydroxylase knockout mice, a model of vitamin D deficiency. Most importantly, we observed no significant hypercalcemia even at supra-physiological concentrations of VDR136. In contrast, calcitriol, a naturally occurring, commercially available vitamin D hormone, caused significant hypercalcemia. Thus, VDR136 represents a novel VDR agonist with significant cardioprotective properties that lack the hypercalcemic effect otherwise common with vitamin D analogs, and may provide a novel therapeutic option for the treatment of cardiac hypertrophy and heart failure.Author Disclosures: J. Lee: None. S.A. Khedkar: Employment; Significant; Imclone. S. Choudhury: None. M. Samad: None. D. Zhang: None. R.I. Thadhani: None. S.A. Karumanchi: None. A.C. Rigby: Employment; Significant; Imclone. P.M. Kang: Research Grant; Significant; Abbott Laboratories.Key Words: Heart failure; Hypertrophy; Vitamins; Calcium; Drugs22966Sorafenib-induced Cardiotoxicity is Mediated by Inhibition of c-kit+ Cardiac Stem CellsCatherine A Makarewich, Jason M Duran, Thomas E Sharp, III, Ronald J Vagnozzi, Remus M Berretta, Hajime Kubo, Thomas Force, Steven R Houser; Temple Univ Sch of Medicine, Philadelphia, PAIntroduction: Tyrosine kinase receptor (RTK) inhibitors have been previously shown to be cardiotoxic in cancer patients with comorbidities, but the mechanism of this cardiotoxicity has not been elucidated. Hypothesis: The multi-kinase inhibitor Sorafenib is known to inhibit several RTKs including c-kit, a receptor found on cardiac progenitor cells in the heart and bone marrow. Patients with comorbidities have pre-existing cardiac damage, and administration of Sorafenib inhibits their cardiac stem cell population, thus preventing myocyte turnover and exacerbating cardiac dysfunction. Methods and Results: Male 12 week old C57BL/6 mice were pretreated with 40 mg/kg/d intraperitoneal Sorafenib (n=20) or vehicle (n=17) for 7 days before receiving myocardial infarction (MI) by coronary artery ligation. This dose had no significant effect on cardiac function before MI. By 1 week post-MI, Sorafenib treatment dramatically exacerbated LV dysfunction measured by echocardiography (ejection fraction = 20.6 ± 3.5 v. 30.6 ± 3.1%; p=0.04) and significantly decreased survival (40.0 v. 72.0%; p=0.02) relative to MI controls. C-kit+ bone marrow stem cells (BMC), c-kit+ cardiac-derived stem cells (CDCs) and adult feline left ventricular myocytes (AFLVMs) were isolated and treated with Sorafenib at 1, 5 and 10uM doses for 12-72 hours in vitro. High concentrations (10 uM) of Sorafenib induced complete necrosis in all cell types. Both 1 and 5 uM treatments potently inhibited proliferation of CDCs and BMCs in a dose-dependent manner in vitro. Low dose Sorafenib (1 uM) produced a time-dependent increase in caspase-3 activation in both cultured BMCs and CDCs suggesting induction of apoptosis/necrosis. Importantly, neither the 1 or 5 uM doses of Sorafenib induced caspase-3 activation in AFLVMs, nor did it cause cell death or otherwise affect contractility or alter calcium transients in vitro. Conclusions: These data show that part of the cardiotoxicity associated with RTK inhibitors, such as Sorafenib, is mediated through inhibition of c-kit+ cardiac progenitor pools. This is the first known report of a proposed mechanism of cardiotoxicity based on a possible inhibition in stem cell-activity (repair-based and/or survival signal-based).Author Disclosures: C.A. Makarewich: None. J.M. Duran: None. T.E. Sharp: None. R.J. Vagnozzi: None. R.M. Berretta: None. H. Kubo: None. T. Force: None. S.R. Houser: None.Key Words: Heart failure; Cardiomyopathy; Drugs; Myocardial infarction22990Cardiac Specific Deletion Of Glycogen Synthase Kinase-3α Attenuates Post Myocardial Infarction-induced Ventricular Remodeling And Preserves Heart FunctionFirdos Ahmad1, Hind Lal1, Ronald J Vagnozzi1, Jibin Zhou1, James R Woodgett2, Erhe Gao1, Thomas Force1; 1Sch of Medicine Temple Univ, Philadelphia, PA, 2Samuel Lunenfeld Rsch Institute, Univ of Toronto, Toronto, CanadaGlycogen synthase kinase (GSK)-3α has been reported to regulate cardiac growth, pathologic hypertrophy, mitochondrial integrity and regulation of contractile function. However, it is essential to better define the role of GSK-3α in the heart using appropriate models since previous studies were performed either by using global knock-in or knock-out strategies, complicating interpretation of the data and limiting understanding of true roles in specific tissues. The goal of this study was to define the role of GSK-3α in the heart using a cardiac-specific conditional GSK-3α KO. For these studies, the α-MHC promoter driving MerCreMer was crossed with the GSK-3αfl/fl mouse. Mice (5-6 months of age) of two genotypes: GSK-3αfl/flCre+Tam+ (KO) and littermate controls (GSK-3αfl/flCre-Tam+(WT), were subjected to LAD ligation (MI). Survival was significantly improved post MI in the KO in comparison to the WT (100% vs 75%, p=0.03). At two weeks post MI, chamber dilation and left ventricular (LV) dysfunction were comparable in KO and WT, though the KO had significantly increased LV mass (p=0.02 at 1 wk, p=0.002 at 2 wks vs WT). Importantly, at four weeks post MI, LV chamber dimension was reduced in the KO [LVID;d(mm) 5.16 ± 0.61 vs 5.97 ± 0.82, p=0.002] and LV function was improved (LVEF% 32.03 ± 9.95 vs 22.3 ± 14.6, p=0.02). The diastolic (p=0.01) and systolic (p=0.007) LV volumes in the KO were also significantly decreased. Our observations are distinctly different from the global GSK-3α KO which had increased post MI mortality and marked cardiac dysfunction. These findings demonstrate that cardiac specific deletion of GSK-3α prevents MI-induced cardiac remodeling and preserves heart function. Moreover, the deletion induces cardiac hypertrophy that seems to be physiological and beneficial for cardiac function. Since inhibition of GSK-3β is also beneficial in this setting, a non-selective inhibitor targeting both isoforms could be a viable therapeutic to control post MI remodeling, preserve heart function and ultimately prevent heart failure.Author Disclosures: F. Ahmad: None. H. Lal: None. R.J. Vagnozzi: None. J. Zhou: None. J.R. Woodgett: None. E. Gao: None. T. Force: Research Grant; Significant; NIH Funding.Key Words: Heart failure; Myocardial infarction; Ventricular remodeling; Heart function tests22807A Clock Gene RORα-mediated Regulation of the Activity of Rho-associated Coiled-coil Protein Kinase 2 (ROCK2) Plays a Key Role in Generating Vascular Intrinsic Circadian Rhythm of Myofilament Ca2+ Sensitivity and Vascular ContractilityToshiro Saito1, Mayumi Hirano1, Tomomi Ide1, Toshihiro Ichiki1, Noriyuki Koibuchi2, Kenji Sunagawa1, Katsuya Hirano1; 1Grad Sch Med Sci, Kyushu Univ, Fukuoka, Japan, 2Gunma Univ Graduate Sch of Medicine, Maebashi, JapanThe occurrence of cardiovascular events shows diurnal variation with a peak in the morning. The circadian change in vascular contractility is one of the mechanisms for this circadian variation. We hypothesize that the vascular intrinsic clock contributes to generating the circadian rhythm of the contractility. However, its underlying mechanism still remains elusive. We addressed this question by using cultured vascular smooth muscle cells (VSMC) and staggerer mutant mice, which lack functional RORα, and identified RORα and ROCK2 that play a key role in generating circadian rhythm of vascular contractility. In VSMC, the level of myosin light chain (MLC) phosphorylation induced by thrombin or endothelin-1 showed an oscillation of a 25.4-hr cycle length with peaks at 36 and 60 hrs and a nadir at 48 hrs after dexamethasone pulse-triggered synchronization of the circadian clock. Pharmacological inhibition or RNAi knockdown of ROCK2, but not MLC kinase, protein kinase C, or ZIP kinase, abolished the rhythm of MLC phosphorylation. The expression of ROCK2 protein and its activity, as assessed by the phosphorylation of MYPT1, oscillated in phase with MLC phosphorylation. The amount of GTP-bound RhoA did not oscillate. The expression of ROCK2 mRNA and RORα protein oscillated in a circadian manner with a peak 4 hrs earlier than the ROCK2 activity. RORα activated the promoter of the ROCK2 gene in luciferase reporter assay, while RORα knockdown abolished the rhythm of ROCK2 expression. ROCK2 expression also oscillated in a circadian manner with a peak at Zeitgeber time (ZT) 0 and a nadir at ZT12 in the aorta of the control, but not staggerer, mice. In the permeabilized aortic ring preparations, the levels of MLC phosphorylation and contraction induced by GTPγS or thromboxane A2 at ZT0 were higher than those seen at ZT12 in the control, but not staggerer, mice. No diurnal change was seen for the Ca2+-dependent contraction in either mouse. We conclude that RORα-induced circadian oscillation of the ROCK2 activity modulates MLC phosphorylation and plays a key role in generating the circadian rhythm of myofilament Ca2+ sensitivity. This rhythm of vascular contractility may underlie the diurnal variation in the occurrence of cardiovascular events.Author Disclosures: T. Saito: None. M. Hirano: None. T. Ide: None. T. Ichiki: None. N. Koibuchi: None. K. Sunagawa: None. K. Hirano: None.Key Words: Smooth muscle; Vascular; Contractility; Signal transductionLate-Breaking Basic Science Poster Session22803Birth Dating of Human Lung Cells by Accelerator Mass SpectrometryMario Ricciardi, Ada Pesapane, Francesca Polverino, Christian Arranto, Giorgia Palano, Hilarie Lam, Ana Castano, Alex Matsuda, Marcello Rota, Annarosa Leri, Ivan Rosas, Mark Perrella, Bruce A Buchholz, Jan Kajstura, Joseph Loscalzo, Piero Anversa; Brigham and Women’s Hosp, Boston, MAThe respiratory units of the distal airway in the human lung are composed of the alveoli and the adjacent capillary structures. Alveoli are defined by type 1 and type 2 epithelial cells, while endothelial cells form a dense capillary network between alveoli. Fibroblasts are distributed at the interface between the alveolar epithelium and endothelial cells (ECs). There is general consensus that type 1 and type 2 pneumocytes, and ECs have limited rene