2015 AHA Late-Breaking Basic Science Abstracts.

Abstract

HomeCirculation ResearchVol. 117, No. 122015 AHA Late-Breaking Basic Science Abstracts Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUB2015 AHA Late-Breaking Basic Science Abstracts Originally published6 Nov 2015https://doi.org/10.1161/RES.0000000000000078Circulation Research. 2015;117:e121–e127Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: January 1, 2015: Previous Version 1 Late-Breaking Basic Science I23060Validation Of Circulating MicroRnas As Biomarkers In Heart Failure In Two Large Independent CohortsAntoni Bayes-Genis1, David Lanfear2, Maurice de Ronde3, Josep Lupón1, Joost Leenders4, Elisabet Zamora1, L. Keoki Williams2, De Antonio Marta5, Zhen Liu4, Koos Zwinderman3, Sara-Joan Pinto-Sietsma3, Yigal Pinto3; 1Universitat Autònoma de Barcelona, Barcelona, Spain; 2Henry Ford Hospital, Detroit, MI; 3Academic Med Cntr, Univ of Amsterdam, Amsterdam, Netherlands; 4Mirnext BV, Amsterdam, Netherlands; 5Germans Trias i Pujol Hosp, Badalona, SpainBackground: Small studies suggested circulating microRNAs (circmiRs) as biomarkers for Heart Failure (HF). However, standardized approaches and quality assessment are not established, and results have been inconsistent, with little replication between studies. We aimed to implement quality standards to enable comparison between cohorts and assess which circmiRs may add prognostic information in HF.Methods: We measured 15 circmiRs in two independent cohorts totaling >2000 subjects. Cohort I (Barcelona) comprised of n=843 chronic HFrEF patients. Cohort II from Detroit comprised n= 1384 chronic HF patients (892 HFrEF, 492 HFpEF). Each sample was measured in duplicate, and normalized to an abundant and stable circmiR (miR-486-5p). Algorithms were installed to define each circmiR measurement as “valid”, “unmeasurable” or “invalid”. This allowed inclusion of valid low-level circmiR measurements while reducing noise from false amplification signals.Results: In general, between 20–40% of measurements were “invalid”, while miR-499a_5p and -208a were “unmeasurable” in the majority of patients in both cohorts. Higher levels of circmiRs-133b, -1254, -622, -208a and -499a_5p were significantly associated with risk of death in both cohorts, with hazard ratios ranging from 1.103 to 1.365 per log increase (p-values 0.001 to 0.05). However, adding these circmiRs to established predictors (age, renal function and NTproBNP) did not further augment the c-stat beyond 0.71 (cohort I) or 0.78 (cohort II).Conclusion: We developed stringent quality assessment for circmiR testing, and for the first time robustly validate the association of circmiRs 208a, -499a_5p, -133b, -1254 and -622 with risk of death in HF patients. However, circmiR levels failed to incrementally improve prognostication offered by current biomarkers, possibly due to the relative high number of invalid measurements. This highlights the shortcomings of current PCR-based technology. Novel technologies under study that improve signal-noise ratios may enhance the prognostic performance of circmiRs.Author Disclosures: A. Bayes-Genis: None. D. Lanfear: Research Grant; Significant; Janssen. M. de Ronde: None. J. Lupón: None. J. Leenders: Employment; Significant; Employed by biomarker company with IP rights related to miRNA. E. Zamora: None. L. Williams: None. D. Marta: None. Z. Liu: Employment; Significant; Employed by biomarker company with IP rights related to miRNA. K. Zwinderman: None. S. Pinto-Sietsma: None. Y. Pinto: Ownership Interest; Modest; owns shares biomarker company with IP rights related to miRNA.Key Words: MicroRNA; Heart Failure; Biomarkers23109Downregulation Of TNF-alpha by In Vivo CRISPR-Cas9 Genome Editing to Treat Cardiac Fibrosis in Diabetic MiceYangxin Li1, Bin Liu2, Bei-bei Lan1, Lian-bo Shao1, Lu-lu Zhang1, Yu Zhang1, Peng-li Xiao1, Xue-yan Jiang3, Qiu-xiong Lin3, Fan Li4, Yong-jian Geng5, Xi-yong Yu6; 1Institute for Cardiovascular Science & Dept of Cardiovascular Surgery, First Affiliated Hosp of Soochow Univ, Suzhou, China; 2Jilin Univ, Changchun, China; 3Guangdong General Hosp, Guangzhou, China; 4Ji Lin Univ, Changchun, China; 5Univ of Texas Health Science Cntr, Houston, TX; 6Guangdong Cardiovascular Institute and Med Rsch Cntr of Guangdong General Hosp, Guangzhou Med Univ, Guangzhou, ChinaIntroduction: Cardiac fibrosis is the leading cause of cardiac dysfunction in patients with diabetes. Currently, there is no effective treatment to prevent the onset of this condition. Inflammation is a hallmark of diabetes associated with heart diseases and TNF-alpha is a key factor involved in inflammation. We tested whether downregulation of TNF-alpha could prevent cardiac fibrosis in diabetic mice.Methods and Results: Diabetes was induced in C57BL/6 mice by injecting streptozotocin (STZ, 55 mg/kg/day) for 5 days. Citrate buffer was injected as control. One week after STZ injection, blood glucose levels significantly increased in diabetic C57BL/6 mice (336 ± 26 mg/dl) compared to control (108 ±21 mg/dl). The increased blood glucose was accompanied by increased cardiac fibrosis (MassionTricrime), and increased TNF-alpha mRNA and protein expression in heart tissue. To assess whether genome editing using a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system can efficiently introduce loss-of-function mutation into the endogenous TNF-alpha gene in vivo, we constructed lentivirus expressing CRISPR-Cas9 and a CRISPR guide RNA targeting TNF-alpha. The lentivirus particles were transplanted into the mouse myocardium by direct intramyocardial injection at the time of initial induction of diabetes. Within 5 days of administration of the lentivirus, the mutagenesis rate of TNF-alpha in the heart was as high as >50% by surveyor assay. No off-target mutagenesis was detected in other tissues such as lung and liver. The CRISPR/Cas9 based knockdown approach resulted in decreased cardiac fibrosis (MassionTricrime), accompanied by reduced expression of TNF -alpha in heart assessed by PCR, Western blot and immunostaining. No significant change of other inflammatory cytokines including IL-6 was observed in the hearts. Furthermore, the CRISPR/Cas9 approach did not alter cell proliferation (BrdU staining) and cell survival (TUNEL assay) in heart.Conclusion: This study demonstrates that inflammation contributes to cardiac fibrosis via TNF-alpha signaling pathway, and suggests that diabetes related cardiac fibrosis may be prevented by downregulation of TNF-alpha via novel CRISPR-Cas9 approach.Author Disclosures: Y. Li: None. B. Liu: None. B. Lan: None. L. Shao: None. L. Zhang: None. Y. Zhang: None. P. Xiao: None. X. Jiang: None. Q. Lin: None. F. Li: None. Y. Geng: None. X. Yu: None.Key Words: CRISPR/Cas9; Inflammation; cardiac fibrosis; TNF-alpha22946Cardiac-Specific Knock-out of the Histone Methyltransferase Smyd1 Leads to Coordinated Downregulation of Energy MetabolismJunko Shibayama, Dane W Barton, Li Wang, Tatiana N Yuzyuk, James Cox, Alexey V Zaitsev, Sarah Franklin; Univ of Utah, Salt Lake City, UTBackground: Global regulatory mechanisms controlling the complex metabolic remodeling which occurs during the onset of heart failure are still poorly understood. However, recent advances have suggested a synergistic link between epigenetic regulation and cellular metabolism, although how this interplay is carried out in the heart is largely unknown. Our recent analyses of chromatin binding proteins differentially regulated during cardiac hypertrophy and failure identified the histone methyltransferase Smyd1. To determine the role of Smyd1 in the adult myocardium we generated inducible, cardiac-specific Smyd1 knockout (Smyd1-KO) mice, which exhibit cellular hypertrophy, chamber remodeling and cardiac dysfunction. In addition, bioinformatics analysis of transcripts differentially expressed in Smyd1-KO heart tissue, before heart function declined, showed that cellular metabolism was the most perturbed biological process in these animals and suggests that Smyd1 may be a key regulator of energy metabolism.Methods and Results: To investigate this hypothesis we carried out metabolomic and gene expression analysis of Smyd1-KO heart tissue to comprehensively characterize the abundance of energetics-related transcripts and metabolites in these animals. Interestingly, our results revealed systemic dysfunction in energy substrate metabolism characterized by downregulation of fatty acid ß-oxidation (observed as a decrease in PPAR-α, carnitine-palmitoyltransferase I, carnitine transporter OCTN2 and myocardial carnitine content [43% reduction]) and branched-chain amino acid (BCAA) oxidation (observed as a 2-fold accumulation of all BCAAs and the decreased expression of PP2Cm, an key activator of BCAA catabolism). In addition, our results identified a dramatic increase in myocardial lactate and alanine (340% and 170% respectively), concomitant with decreased expression of pyruvate dehydrogenase E1 β, indicative of glycolytic impairment.Conclusion: Overall, this study identifies a novel role for Smyd1 in regulating energy metabolism in the heart and provides key insights into the epigenetic mechanisms modulating metabolic disorders such as heart failure.Author Disclosures: J. Shibayama: None. D.W. Barton: None. L. Wang: None. T.N. Yuzyuk: None. J. Cox: None. A.V. Zaitsev: None. S. Franklin: None.Key Words: Cardiac hypertrophy/failure; Smyd1 (histone methyltransferase); Epigenetics; Cardiac energetics; Metabolomics/Transcriptomics23147Circulating miR-1 And miR-133b Correlate With Subclinical Myocardial Injury In Breast Cancer Patients Under Doxorubicin TreatmentVagner Oliveira-Carvalho, Ludmila R Ferreira, Danielle P Borges, Silvia M Ayub-Ferreira, Mônica S Ávila, Sara M Brandão, Fátima Cruz, Edécio Cunha-Neto, Edimar A Bocchi; Instituto do Coração, São Paulo, BrazilIntroduction: Myocardial injury is one of the major concerns when doxorubicin (DOX) is chronically administered for several weeks and may lead to cardiomyopathy and heart failure. Recently, circulating microRNAs (c-miRNAs) have been suggested as potential biomarkers of myocardial injury and drug-induced cardiotoxicity. However, the potential of c-miRNAs as biomarkers of DOX-induced cardiotoxicity in a clinical setting was not assessed. Therefore, the aim of this study is to evaluate the cardiotoxic effects of DOX on the circulating levels of miR-1, miR-133b, miR-146a, miR-208a, miR-208b and miR-423-5p in breast cancer patients.Methods: In brief, 59 female patients (50,02±8,64 age) received 4 cycles of chemotherapy with cumulative doses of 60 mg/m2 DOX during 12 weeks. Cardiac troponin I (cTnI), LVEF and c-miRNAs were measured before the treatment and every 3 weeks after DOX administration. Results: miR-208a and miR-208b were undetectable in plasma even after the maximum dose of DOX. Circulating levels of miR-1, miR-133b, miR-146a and miR-423-5p were increased along the treatment reaching its peak at the third cycle (increase of 18,6-fold, 11,51-fold, 10,56-fold and 12,09-fold respectively; P<0,001) and earlier than cTnI (fourth cycle). For further analysis, patients with 2 SD above the mean were grouped as “High cTnI” and compared to the others (“control”). High cTnI group (n= 7) showed a cTnI increase from 6.0±1.6 to 134.0±10.52 whereas from 6.4±0.2 to 31±2.8 pg/ml in the control group (n= 52; P<0,001). No changes in LVEF were observed within or between the groups. Despite all miRNAs analyzed had been upregulated during the treatment, only miR-1 and miR-133b (muscle-specific miRNAs) were differently expressed in high cTnI patients showing a positive correlation with cTnI (P<0,05). Respectively, miR-1 and miR-133b were increased by 5,05-fold and 4,46-fold (P<0,05) in high cTnI patients when compared with controls.Conclusion: Circulating miR-1 and miR-133b were upregulated by DOX treatment and correlate with subtle myocardial injury in a “normal heart”. These findings may lead to the development of biomarkers to monitor DOX-induced subclinical myocardial injury avoiding its progression to irreversible cardiomyopathy and heart failure.Author Disclosures: V. Oliveira-Carvalho: None. L.R. Ferreira: None. D.P. Borges: None. S.M. Ayub-Ferreira: None. M.S. Ávila: None. S.M. Brandão: None. F. Cruz: None. E. Cunha-Neto: None. E.A. Bocchi: Research Grant; Modest; Servier. Other Research Support; Modest; Baldacci. Speakers Bureau; Modest; Servier, Novartis, Baldacci. Consultant/Advisory Board; Modest; Sevier, Novartis.Key Words: microRNA; Cardiotoxicity; Doxorubicin; Myocardial Injury; Breast CancerLate-Breaking Basic Science II22915A Novel TRPV4-dependent Mechanotranscription Pathway Regulates Cardiac Fibrosis Following Pressure-OverloadRavi K Adapala1, Holly Cappelli1, Vahagn A Ohanyan1, Roslin Thoppil1, Jordan Luli1, Sailaja Paruchuri2, William M Chilian1, J. Gary Meszaros1, Charles Thodeti1; 1Northeast Ohio Med Univ, Rootstown, OH, 2Univ of Akron, Akron, OHCardiac fibroblast (CF) differentiation into highly contractile and hypersecretory myofibroblasts (myoFibs) is critical for reparative fibrosis following myocardial injury/insult. However, excessive and remote area fibrosis by myoFibs can lead to cardiac dysfunction and eventual heart failure. Recently, we have shown that a mechanosensitive ion channel TRPV4 mediates CF differentiation to myoFib in-vitro. In the present study, we investigated the underlying molecular mechanism and the physiological role of TRPV4 during cardiac remodeling following pressure overload (transverse aortic constriction, TAC), in wild type (WT) and TRPV4 knockout (TRPV4KO) mice. We found that TRPV4KO mice exhibited not only improved survival rates compared to WT, but cardiac function analysis showed preserved ejection fraction and fractional shortening in TRPV4 null mice, post-TAC surgeries. Importantly, we found that TAC induced both interstitial and perivascular fibrosis in WT hearts, which was significantly reduced in TRPV4KO hearts. To understand the molecular mechanism, we isolated CFs from WT and TRPV4KO mouse hearts (mCFs). In vitro, we found that TGF-β1-induced differentiation was completely attenuated in TRPV4KO mCFs compared to WT. Further, TGF-β1 treatment induced activation of RhoA in WT mCFs, which was inhibited by pre-treatment with TRPV4 antagonist, AB159908, suggesting that Rho is downstream of TRPV4 in CF differentiation to myoFibs. Further, our recent results found that both TGF-β1 and pharmacological TRPV4 activator GSK1016790A induced the activation of myocardin-related transcription factor-A (MRTF-A) (nuclear translocation), that was abolished when TRPV4 was inhibited. Furthermore, we found that both TGF-β1 and GSK1016790A induced α-SMA and col1a promoter activities indicating CF differentiation. Finally, we found while pharmacological activation of MRTF-A increased, MRTF-A inhibition attenuated TGF-β1-induced CF differentiation. Taken together, these findings suggest that TRPV4 channels mediate cardiac fibrosis through a mechanosensitive transcriptional (Rho/MRTF-A) pathway, and targeting TRPV4 could offer novel therapeutics for treating cardiac fibrosis.Author Disclosures: R.K. Adapala: None. H. Cappelli: None. V.A. Ohanyan: None. R. Thoppil: None. J. Luli: None. S. Paruchuri: None. W.M. Chilian: Consultant/Advisory Board; Modest; Angionetics, Inc.. Research Grant; Significant; NIH RO1 grant. J. Meszaros: None. C. Thodeti: None.Key Words: TRPV4; cardiac fibroblast; myofibroblast; mechanical signaling; fibrosis22961Physiologic Mitochondrial Fragmentation is a Cardiac Adaptation to Increased Energy DemandMichael Coronado1, Giovanni Fajardo1, Kim Nguyen1, Mingming Zhao1, Kristina Bezold Kooiker1, Gwanghyun Jung1, Dong-Qing Hu1, Sushma Reddy1, Erik Sandoval1, Aleksandr Stotland2, Roberta Gottlieb2, Daniel Bernstein1; 1Stanford Univ, Stanford, CA,; 2Cedars-Sinai Med Cntr, Los Angeles, CA,Current paradigms hold that, in the heart, mitochondrial fission and fragmentation are the result of pathologic stresses such as ischemia, are an indicator of poor mitochondrial health, and lead to mitophagy and cell death. However, recent studies demonstrate that inhibiting fission also results in cardiac impairment, suggesting that fission is important for maintaining normal mitochondrial homeostasis. As these studies have relied on genetic manipulation of dynamic regulators, the role of fission in normal cardiac physiology is still unclear. In this study, we identify a novel role for mitochondrial fragmentation, as a normal physiologic adaptation to meet the energetic demands of exercise. During acute exercise in mice, there is significant “physiologic” mitochondrial fragmentation in the heart, demonstrated by a 30% increase in mitochondrial number and a 20% decrease in area (electron microscopy). Whereas pathologic fragmentation leads to impaired respiration, physiologic fragmentation increases both basal and maximal respiration by 20% (seahorse oximetry). Similar to pathologic fragmentation, physiologic fragmentation is induced by translocation of Drp1 to the mitochondria; however, unlike pathologic fragmentation, mitochondrial membrane potential and reactive oxygen species are maintained and regulators of mitophagy (PINK1, Parkin, LC3) are downregulated. Inhibition of Drp1 during exercise (using either the Drp1-Fis1 inhibitor P110 or the Drp1 GTPase inhibitor mDivi) prevents mitochondrial fragmentation in the heart, increases glycolytic flux and results in a 40% decrease in exercise capacity, demonstrating the requirement for physiologic mitochondrial fragmentation to meet the energetic demands of exercise. In summary, we have shown that cardiac mitochondrial fragmentation is not limited to pathologic states and can be a component of normal physiological regulation of mitochondrial function during stresses such as exercise. Whether physiologic fragmentation can be harnessed as a treatment of cardiovascular disease remains to be determined, although some of the cardiovascular benefit of exercise could be mediated by such a mechanism.Author Disclosures: M. Coronado: None. G. Fajardo: None. K. Nguyen: None. M. Zhao: None. K. Bezold Kooiker: None. G. Jung: None. D. Hu: None. S. Reddy: None. E. Sandoval: None. A. Stotland: None. R. Gottlieb: None. D. Bernstein: None.Key Words: Mitochondria; Adrenergic; Physical activity and exercise; Mitochondrial energetics; Muscle, cardiac23179DNA-repair In Cardiomyocytes Is Critical For Maintaining Cardiac FunctionMartine de Boer1, Yanti Octavia1, Marion G de Kleijnen1, Bibi S van Thiel2, Yanto Ridwan3, Maaike te Lintel Hekkert1, Ingrid van der Pluijm4, Jeroen Essers5, Jan H Hoeijmakers6, Dirk J Duncker1; 1Div of Experimental Cardiology, Dept of Cardiology, Thoraxcenter, Cardiovascular Rsch Sch COEUR, Erasmus MC, Univ Med Cntr Rotterdam, Rotterdam, Netherlands; 2Dept of Genetics; Dept of Vascular Surgery, Div of Vascular Medicine and Pharmacology; Dept of Internal Medicine, Erasmus MC, Univ Med Cntr Rotterdam, Rotterdam, Netherlands; 3Dept of Genetic; Div of Vascular Medicine and Pharmacology, Dept of Internal Medicine, Erasmus MC, Univ Med Cntr Rotterdam, Rotterdam, Netherlands; 4Dept of Genetics; Dept of Vascular Surgery,Erasmus MC, Univ Med Cntr Rotterdam, Rotterdam, Netherlands; 5Dept of Genetics; Dept of Vascular Surgery; Dept of Radiation Oncology, Erasmus MC, Univ Med Cntr Rotterdam, Rotterdam, Netherlands; 6Dept of Genetics, Erasmus MC, Univ Med Cntr Rotterdam, Rotterdam, NetherlandsAims: DNA in every cell is continuously damaged and DNA repair systems are essential for protection from DNA damage induced cancer and aging- related diseases. Here we studied the role of DNA repair in cardiomyocytes in maintaining normal cardiac function. DNA repair-deficient full body Xpg-/- and cardiomyocyte-specific Xpgc/- (αMHC-Xpg) mice were used to study left ventricular (LV) geometry and function.Methods: Xpg-/- (n=18), αMHC-Xpg (n=14) and control wildtype (wt, n=14) mice were sacrificed at the age of 16 wks. LV geometry and function were measured and hypertrophy marker genes were determined by qPCR. Superoxide (O2-) production and NOX activity were studied using lucigenin-enhanced chemiluminiscence. NO synthase (NOS) and NADPH Oxidase (NOX) - dependent O2- were assessed with L-NAME and VAS2870. In αMHC-Xpg and wt mice (both n=4), molecular imaging was performed to determine apoptosis in the in vivo heart using near infrared fluorescent Annexin V probe.Results: Xpg-/- mice showed reduced growth, followed by body weight loss and shortened lifespan (18 wks). αMHC-Xpg mice exhibited normal growth and body weight gain, but also reduced lifespan (28 wks). At 16 wks, LV function had deteriorated in both Xpg-/- and αMHC-Xpg compared to wt (Table 1). Total and NOS-dependent O2- production was only increased in Xpg-/-. In the presence of NADPH, NOX activity was elevated in both groups, but NOX-dependent O2- generation was higher only in Xpg-/-. The relative RNA expression level of atrial natriuretic peptide was increased in both groups, but particularly in αMHC-Xpg. Moreover, αMHC-Xpg showed a marked increase in LV end-diastolic lumen diameter and displayed a marked increase in in vivo cardiac apoptosis (27±2pmol vs. 6±1pmol in wt; p<0.05).Download figureDownload PowerPointConclusion: Mice with (cardiomyocyte-restricted) loss of DNA-repair protein XPG display a heart failure phenotype, demonstrating that intact DNA repair in cardiomyocytes is critical for maintaining normal cardiac function.Author Disclosures: M. de Boer: None. Y. Octavia: None. M.G.J. de Kleijnen: None. B.S. van Thiel: None. Y. Ridwan: None. M. te Lintel Hekkert: None. I. van der Pluijm: None. J. Essers: None. J.H. Hoeijmakers: None. D.J. Duncker: None.Key Words: cardiac function; DNA repair; XPG; oxidative stress; mice23289Dysregulation of the Nonmyocyte Microenvironment Promotes Rapid Load-Induced Heart Failure via Activation of the ERK Signaling CascadeRosanne Rouf, Elena Gallo-Macfarlane, Eiki Takimoto, Nori Koitabashi, Peter Rainer, Djahida Bedja, Elizabeth Gerber, Julia Bindman, Christopher Schiefer, Karen Miller, Guangshuo Zhu, Rahul Chaudhary, Daniel Judge, David Kass, Harry Dietz III; Johns Hopkins Sch of Medicine, Baltimore, MDBackground: Little is known about how perturbations in the ECM microenvironment regulate nonmyocyte function. This study was designed to assess whether deficiency of fibrillin-1, a ubiquitous ECM molecule that is expressed primarily in fibroblast-like cells, renders the heart more vulnerable to mechanical stress.Methods and Results: Baseline cardiac structure and function were indistinguishable between WT and fibrillin-1 deficient (Fbn1C1039G/+) mice. However, after only 1 week of pressure overload induced by transverse aortic constriction (TAC), Fbn1C1039G/+ hearts showed dramatic dilation and dysfunction which progressed in the ensuing 3 weeks. Fbn1C1039G/+:TAC heart mass was profoundly increased compared with WT:TAC (14.1±1.6 vs 6.2±0.1 mg/g, p<0.05) as were myocyte area (224±3 vs 206±4μm2, p<0.05) and fibrosis (fold change to SHAM, 3.3±0.2 vs 1.3±0.3, p<0.05). Though Fbn1 expression increased in Fbn1C1039G/+:TAC and WT:TAC hearts, fibrillin-1 deposition in the ECM microenvironment increased only in WT:TAC which notably had compensated function at study end. Intriguingly, in Fbn1C1039G/+:TAC hearts, pERK1/2 was enhanced selectively in vimentin-positive nonmyocytes, whereas enhanced pSmad2 was found in both nonmyocyte and myocyte cell compartments. Remarkably, treatment pre- and post-TAC with a selective MEK inhibitor of ERK1/2 phosphorylation reversed dysfunction despite chronic load. Furthermore, not only was pERK1/2 normalized but also pSmad2 was normalized in both the nonmyocyte and myocyte cell compartments, fibrosis was reduced, and myocyte area returned to normal, implicating Erk-mediated TGFβ-dependent autocrine/paracrine pathways at play. Lastly, in the WT heart, while Fbn1 expression and deposition were increased during compensated hypertrophy, Fbn1 was significantly decreased in the transition to decompensated function which correlated with increased TGFβ signaling and expression of TGFβ targets.Conclusion: Fibrillin-1 deposition in the nonmyocyte microenvironment is an important cardioprotective adaptation that opposes decompensated heart failure. These findings suggest that manipulation of cell-specific microenvironments may represent a therapeutic target for ameliorating heart failure.Author Disclosures: R. Rouf: None. E. Gallo-Macfarlane: None. E. Takimoto: None. N. Koitabashi: None. P. Rainer: None. D. Bedja: None. E. Gerber: None. J. Bindman: None. C. Schiefer: None. K. Miller: None. G. Zhu: None. R. Chaudhary: None. D. Judge: None. D. Kass: None. H. Dietz: None.Key Words: fibrillin-1; microenvironment; nonmyocyte; MAPK; transforming growth factor betaLate-Breaking Basic Science Posters22950Novel Role Of MicroRNA-532 In Vascular FunctionSadie Slater, Marketa Rodova, Iker Rodriguez-Arabaolaza, Valeria Alvino, Eva Jover Garcia, Federica Riu, Paolo Madeddu; Univ of Bristol, Bristol, United KingdomRecent evidence indicates the importance of microRNAs in the control of vascular homeostasis. However, little is known about the expression of microRNAs in human perivascular cells, namely microvascular and adventitial pericytes. The latter are attracting much attention owing to their potent pro-healing activity, which reportedly involves microRNA-132. Here, we focus on microRNA-532 as a novel regulator of pericyte function. Adventitial pericytes were isolated from vein leftovers of coronary artery bypass graft surgery (n=7 patients). Using a miRCURY Universal RT microRNA PCR Human array, we found 175 microRNAs expressed in all samples, with 19 of them being differentially regulated by hypoxia. By qPCR, we confirmed that microRNA-532 is downregulated by hypoxia (P<0.0001). We next used a Taqman anti-miR inhibitor to reduce microRNA-532 expression to levels similar to those seen under hypoxia. Inhibition of microRNA-532 resulted in enhanced migratory activity as assessed by scratch assays (1.5-fold, P<0.05), without affecting pericyte proliferation or apoptosis. In vitro Matrigel assay showed microRNA-532-inhibited pericytes have reduced capacity to promote network formation by HUVECs, which was associated with prevalent localization of Dil-labelled pericytes around network branches rather than notches. Furthermore, conditioned media from microRNA-532-inhibited pericytes increase the permeability of HUVEC monolayers. Among microRNA-532 candidate targets, we found leptin to be upregulated following pericyte exposure to hypoxia or microRNA-532 silencing. Studies using leptin-silencing confirmed contribution of this hormone in induction of vascular permeability by hypoxic pericytes. On the other hand, angiopoietin-1, a potent vascular stabilizer produced by pericytes, was remarkably downregulated following microRNA-532 silencing. This effect is indirect as angiopoietin-1 is not a direct target microRNA-532. In summary, microRNA-532 is modulated by hypoxia and involved in the control of pericyte motility, angiogenic activity and modulation of vascular permeability. These data reveal a previously unforeseen role of microRNA-532 in vascular biology.Author Disclosures: S. Slater: None. M. Rodova: None. I. Rodriguez-Arabaolaza: None. V. Alvino: None. E. Jover Garcia: None. F. Riu: None. P. Madeddu: None.Key Words: microRNA; Vascular; Hypoxia23164Functional Consequences of a Rare TBX5 Variant In Familial Brugada SyndromeKevin R Bersell1, Charles C Hong1, Elijah R Behr2, Evmorfia Petropoulou2, Yalda Jamshidi2, Quinn Wells1, Prince Kannankeril1, Dan M Roden1; 1Vanderbilt Univ Sch of Medicine, Nashville, TN; 2St George’s Univ of London, London, United KingdomIntroduction: The Brugada syndrome (BrS) is a channelopathy with a distinctive ECG pattern reflecting decreased sodium current and increased risk of sudden death. Loss of function variants in the cardiac sodium channel gene, SCN5A, account for ~20% of cases. Reduced channel expression has been proposed as a mechanism in other cases of BrS, but functional experiments that directly link regulatory variants to altered ionic current in BrS or other channelopathies have not been reported.Methods and Results: We identified a family with multiple individuals displaying the BrS ECG, but no rare coding region variants in SCN5A. However, Sanger sequencing of other candidate genes identified a previously unreported non-synonymous variant of unknown significance (G145R) in TBX5; Tbx5 haploinsufficiency in mice produces multiple phenotypes, including reduced Scn5a expression. In TBX5-driven reporter assays, TBX5-G145R reduced luciferase expression compared to wild-type protein. To further establish the functional consequences of TBX5-G145R, we studied cardiomyocytes differentiated from induced pluripotent stem cells (iPSC-CMs) derived from mutation carriers in the family. In BrS iPSC-CMs studied 35 days after cardiomyocyte induction, SCN5A transcript abundance was decreased compared to multiple control lines. Peak sodium current at -30 mV (from a holding potential of -100 mV) was -97.1±36.5 pA/pF (n=8) in the BrS myocytes compared to -211±36.0 pA/pF (n=13 from two unique donors; p<0.05) in controls (Figure).Download figureDownload PowerPointConclusion: These data provide strong evidence that TBX5-G145R causes the Brugada syndrome phenotype in this family by reducing cardiac sodium channel expression. The development of iPSC-CMs has allowed us for the first time to directly link altered ion channel gene expression to a channelopathy, and this approach will therefore enable definition of the role of regulatory elements as causes and modulators of cardiac arr