channel conductance. These results are consistent with the time course of thecurrent changes seen with NADH/NADþ.

EX VIVO AND IN VIVO ADMINISTRATION OF FLUORESCENT CNA35-PROTEINSPECIFICALLY MARKS CARDIAC FIBROSISS. de Jong1, L. van Middendorp2, R.H.A. Hermans2, J.M.T. de Bakker1,M.F.A. Bierhuizen1, F.W. Prinzen2, H.V.M. van Rijen1, M.A. Vos1,M.A.M. van Zandvoort21University Medical Center Utrecht, Utrecht, Netherlands, 2MaastrichtUniversity Medical Center, Maastricht, Netherlands.

Background: Cardiac fibrosis has severe adverse effects on systolic anddiastolic function and increases the risk of arrhythmias. To detect cardiacfibrosis, the development of highly specific and preferably noninvasivemethods is desired. In this study, the potential of labeled CNA35, a proteinthat specifically binds to vascular collagen, is investigated as a specificmarker of cardiac fibrosis. Picrosirius red (PSR) staining served as referencefor cardiac fibrosis detection.Methods: Fluorescently labeled CNA35 (2.5 mM) was applied to frozen tissuesections of dog myocardium (n ¼ 36). After CNA35 quantification, sectionswere histologically examined with PSR and compared to CNA35. Furthermore,fluorescently labeled CNA35 (2 � 38 mM) was administered intravenously inmice (n¼ 8). Hearts were isolated, and CNA35 labeling was examined in frozentissue sections (n ¼ 34). Serial sections were examined with PSR.Results: CNA35 application on frozen canine myocardium shows specificCNA35 binding to collagen. CNA35 labeling highly correlates with PSR(r ¼ 0.98, P o.001). After in vivo administration in mice, myocardialCNA35 labeling is clearly observed, suggesting that labeled CNA35 is ableto penetrate the cardiac endothelium. Strong correlation is observed betweenCNA35 and PSR (r ¼ 0.91, P o.001; Figure).Conclusions: CNA35 specifically binds to cardiac collagen when applied tofrozen sections and after in vivo application of CNA35. Strong correlation hasbeen observed between CNA35 and PSR. These data indicate that CNA35 isuseful for quantification and localization of myocardial fibrosis and warrantsfurther research into its use for monitoring cardiac fibrosis in vivo.

MISSENSE MUTATIONS IN PLAKOPHILIN-2 CAN CAUSE BRUGADASYNDROME PHENOTYPE BY DECREASING SODIUM CURRENT AND Nav1.5MEMBRANE LOCALIZATIONM. Cerrone1, X. Lin1, M. Zhang1, E. Agullo-Pascual1, A. Pfenniger1,H. Chkourko Gusky1, V. Novelli2, C. Kim3, T. Tirasawadischai3,

D.P. Judge4, E. Rothenberg1, H.V. Chen3, C. Napolitano2, S.G. Priori2,M. Delmar11NYU School of Medicine, New York, NY, 2IRCCS Fondazione Maugeri,Pavia, Italy, 3Sanford-Burnham Medical Research Institute, La Jolla, CA,4Johns Hopkins University School of Medicine, Baltimore, MD.

Background: Brugada syndrome (BrS) is associated with loss of sodiumchannel function. Previous studies showed features consistent with sodiumcurrent (INa) deficit in patients carrying desmosomal mutations, diagnosedwith arrhythmogenic cardiomyopathy ([AC]; or arrhythmogenic rightventricular cardiomyopathy [ARVC]). Experimental models showed corre-lation between loss of expression of desmosomal protein plakophilin-2(PKP2) and reduced INa. We hypothesized that PKP2 variants that reduce INacould yield a BrS phenotype, even without cardiomyopathic features of AC.Methods and Results: We searched for PKP2 variants in genomic DNA of200 patients with BrS diagnosis, no signs of AC, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L, and MOG1. We identified 5 casesof single amino acid substitutions. One (Q62K) was previously described inAC patients as a variant of unknown significance; 4 were unreported. In afamily with multiple cases of syncope and/or suspect ECG, novel variantR635Q cosegregated with the phenotype in all affected relatives and wasabsent in the nonaffected ones. Mutations were tested in HL-1–derived cellsendogenously expressing Nav1.5 but made deficient in PKP2 (PKP2-KD).Loss of PKP2 caused decreased INa and Nav1.5 at site of cell contact. Thesedeficits were restored by transfection of wild-type PKP2 (PKP2-WT) but notof BrS-related PKP2 mutants. Similar results were obtained when cells werecotransfected with PKP2-WT and the BrS-related PKP2 variants, to mimicheterozygosity. Human-induced pluripotent stem cell cardiomyocytes(hiPSC-CMs) from a patient with PKP2 deficit showed drastically reducedINa. The deficit was restored by transfection of WT but not BrS-related PKP2variant R635Q. Superresolution microscopy in murine PKP2-deficientcardiomyocytes related INa deficiency to reduced number of channels at theintercalated disk and increased separation of microtubules from the cell end.Conclusions: This is the first systematic retrospective analysis of a patientgroup to define the coexistence of sodium channelopathy and genetic PKP2variations. PKP2 mutations may be a molecular substrate leading to thediagnosis of BrS.

GENOME-WIDE ASSOCIATION ANALYSIS IDENTIFIES 3 COMMON VARIANTSPREDISPOSING TO BRUGADA SYNDROME, A RARE DISEASE WITH HIGHRISK OF SUDDEN CARDIAC DEATHJ. Barc1, C. Bezzina1, Y. Mizusawa1, C. Remme1, J. Gourraud2, A. Verkerk1,P. Schwartz3, P. Guicheney4, C. Antzelevitch5, E. Schulze-Bahr6, E. Behr7,J. Tfelt-Hanson8, S. Kaab9, H. Watanabe10, M. Horie11, N. Makita12,W. Shimizu13, D. Roden14, V. Christoffels1, M. Gessler15, A. Wilde1,V. Probst2, J. Schott2, C. Dina2, R. Redon21Heart Failure Research Center, Amsterdam, Netherlands, 2l’Institut duThorax, Université de Nantes, CHU Nantes, Nantes, France, 3University ofPavia, Pavia, Italy, 4Faculté de Médecine Pierre et Marie Curie, Paris,France, 5Masonic Medical Research Laboratory, Utica, NY, 6Institute forGenetics of Heart Diseases (IfGH), Munster, Germany, 7St. George'sUniversity of London, London, United Kingdom, 8University ofCopenhagen, Copenhagen, Denmark, 9University Hospital Munich,Munich, Germany, 10Niigata University Graduate School of Medical andDental Sciences, Niigata, Japan, 11Shiga University of Medical Science,Otsu, Japan, 12Nagasaki University, Nagasaki, Japan, 13National Cerebraland Cardiovascular Center, Suita, Japan, 14Vanderbilt University School ofMedicine, Nashville, TN, 15University of Wuerzburg, Wuerzburg, Germany

Background: The Brugada syndrome (BrS) is considered a rare mendeliandisorder with autosomal dominant transmission. BrS is associated with anincreased risk of sudden cardiac death and specific ECG features consistingof ST-segment elevation in the right precordial leads. Loss-of-functionmutations in SCN5A, encoding the pore-forming subunit of the cardiacsodium channel (Nav1.5), are identified in �20% of patients. However,studies in families harboring mutations in SCN5A have demonstrated lowdisease penetrance and, in some instances, absence of the familial SCN5Amutation in some affected members. These observations suggest a morecomplex inheritance model.

1743