channel conductance. These results are consistent with the time course of the current changes seen with NADH/NAD þ . EX VIVO AND IN VIVO ADMINISTRATION OF FLUORESCENT CNA35-PROTEIN SPECIFICALLY MARKS CARDIAC FIBROSIS S. de Jong 1 , L. van Middendorp 2 , R.H.A. Hermans 2 , J.M.T. de Bakker 1 , M.F.A. Bierhuizen 1 , F.W. Prinzen 2 , H.V.M. van Rijen 1 , M.A. Vos 1 , M.A.M. van Zandvoort 2 1 University Medical Center Utrecht, Utrecht, Netherlands, 2 Maastricht University Medical Center, Maastricht, Netherlands. Background: Cardiac fibrosis has severe adverse effects on systolic and diastolic function and increases the risk of arrhythmias. To detect cardiac fibrosis, the development of highly specific and preferably noninvasive methods is desired. In this study, the potential of labeled CNA35, a protein that specifically binds to vascular collagen, is investigated as a specific marker of cardiac fibrosis. Picrosirius red (PSR) staining served as reference for cardiac fibrosis detection. Methods: Fluorescently labeled CNA35 (2.5 mM) was applied to frozen tissue sections of dog myocardium (n ¼ 36). After CNA35 quantification, sections were histologically examined with PSR and compared to CNA35. Furthermore, fluorescently labeled CNA35 (2  38 mM) was administered intravenously in mice (n ¼ 8). Hearts were isolated, and CNA35 labeling was examined in frozen tissue sections (n ¼ 34). Serial sections were examined with PSR. Results: CNA35 application on frozen canine myocardium shows specific CNA35 binding to collagen. CNA35 labeling highly correlates with PSR (r ¼ 0.98, P o.001). After in vivo administration in mice, myocardial CNA35 labeling is clearly observed, suggesting that labeled CNA35 is able to penetrate the cardiac endothelium. Strong correlation is observed between CNA35 and PSR (r ¼ 0.91, P o.001; Figure). Conclusions: CNA35 specifically binds to cardiac collagen when applied to frozen sections and after in vivo application of CNA35. Strong correlation has been observed between CNA35 and PSR. These data indicate that CNA35 is useful for quantification and localization of myocardial fibrosis and warrants further research into its use for monitoring cardiac fibrosis in vivo. MISSENSE MUTATIONS IN PLAKOPHILIN-2 CAN CAUSE BRUGADA SYNDROME PHENOTYPE BY DECREASING SODIUM CURRENT AND Na v 1.5 MEMBRANE LOCALIZATION M. Cerrone 1 , X. Lin 1 , M. Zhang 1 , E. Agullo-Pascual 1 , A. Pfenniger 1 , H. Chkourko Gusky 1 , V. Novelli 2 , C. Kim 3 , T. Tirasawadischai 3 , D.P. Judge 4 , E. Rothenberg 1 , H.V. Chen 3 , C. Napolitano 2 , S.G. Priori 2 , M. Delmar 1 1 NYU School of Medicine, New York, NY, 2 IRCCS Fondazione Maugeri, Pavia, Italy, 3 Sanford-Burnham Medical Research Institute, La Jolla, CA, 4 Johns Hopkins University School of Medicine, Baltimore, MD. Background: Brugada syndrome (BrS) is associated with loss of sodium channel function. Previous studies showed features consistent with sodium current (I Na ) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy ([AC]; or arrhythmogenic right ventricular cardiomyopathy [ARVC]). Experimental models showed corre- lation between loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced I Na . We hypothesized that PKP2 variants that reduce I Na could yield a BrS phenotype, even without cardiomyopathic features of AC. Methods and Results: We searched for PKP2 variants in genomic DNA of 200 patients with BrS diagnosis, no signs of AC, and no mutations in BrS- related genes SCN5A, CACNa1c, GPD1L, and MOG1. We identified 5 cases of single amino acid substitutions. One (Q62K) was previously described in AC patients as a variant of unknown significance; 4 were unreported. In a family with multiple cases of syncope and/or suspect ECG, novel variant R635Q cosegregated with the phenotype in all affected relatives and was absent in the nonaffected ones. Mutations were tested in HL-1–derived cells endogenously expressing Na v 1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased I Na and Na v 1.5 at site of cell contact. These deficits were restored by transfection of wild-type PKP2 (PKP2-WT) but not of BrS-related PKP2 mutants. Similar results were obtained when cells were cotransfected with PKP2-WT and the BrS-related PKP2 variants, to mimic heterozygosity. Human-induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from a patient with PKP2 deficit showed drastically reduced I Na . The deficit was restored by transfection of WT but not BrS-related PKP2 variant R635Q. Superresolution microscopy in murine PKP2-deficient cardiomyocytes related I Na deficiency to reduced number of channels at the intercalated disk and increased separation of microtubules from the cell end. Conclusions: This is the first systematic retrospective analysis of a patient group to define the coexistence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS. GENOME-WIDE ASSOCIATION ANALYSIS IDENTIFIES 3 COMMON VARIANTS PREDISPOSING TO BRUGADA SYNDROME, A RARE DISEASE WITH HIGH RISK OF SUDDEN CARDIAC DEATH J. Barc 1 , C. Bezzina 1 , Y. Mizusawa 1 , C. Remme 1 , J. Gourraud 2 , A. Verkerk 1 , P. Schwartz 3 , P. Guicheney 4 , C. Antzelevitch 5 , E. Schulze-Bahr 6 , E. Behr 7 , J. Tfelt-Hanson 8 , S. Kaab 9 , H. Watanabe 10 , M. Horie 11 , N. Makita 12 , W. Shimizu 13 , D. Roden 14 , V. Christoffels 1 , M. Gessler 15 , A. Wilde 1 , V. Probst 2 , J. Schott 2 , C. Dina 2 , R. Redon 2 1 Heart Failure Research Center, Amsterdam, Netherlands, 2 l’Institut du Thorax, Université de Nantes, CHU Nantes, Nantes, France, 3 University of Pavia, Pavia, Italy, 4 Faculté de Médecine Pierre et Marie Curie, Paris, France, 5 Masonic Medical Research Laboratory, Utica, NY, 6 Institute for Genetics of Heart Diseases (IfGH), Munster, Germany, 7 St. George's University of London, London, United Kingdom, 8 University of Copenhagen, Copenhagen, Denmark, 9 University Hospital Munich, Munich, Germany, 10 Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan, 11 Shiga University of Medical Science, Otsu, Japan, 12 Nagasaki University, Nagasaki, Japan, 13 National Cerebral and Cardiovascular Center, Suita, Japan, 14 Vanderbilt University School of Medicine, Nashville, TN, 15 University of Wuerzburg, Wuerzburg, Germany Background: The Brugada syndrome (BrS) is considered a rare mendelian disorder with autosomal dominant transmission. BrS is associated with an increased risk of sudden cardiac death and specific ECG features consisting of ST-segment elevation in the right precordial leads. Loss-of-function mutations in SCN5A, encoding the pore-forming subunit of the cardiac sodium channel (Na v 1.5), are identified in  20% of patients. However, studies in families harboring mutations in SCN5A have demonstrated low disease penetrance and, in some instances, absence of the familial SCN5A mutation in some affected members. These observations suggest a more complex inheritance model. 1743